HU230463B1 - Control system for immunoprecipitation studies - Google Patents

Abstract

The, present invention provides for uses of a nanoparticle e.g. a virus or virion, preferably a bacteriophage, for providing control in an immunoprecipitation reaction with a recognition molecule, wherein an epitope peptide is displayed oh said naripparticle, e.g. by said virus or virion and recognized by said recognition molecule in the immunoprecipitation reaction. The nanoparticle carries a nucleic acid comprising a sequence encoding said epitope. Preferably, the immunoprecipitation is chromatin immunoprecipitation (ChIP).

Description

(54) Control system for immunoprecipitation assays (57) Extract

The subject of the invention is the use of nanoparticles, viruses, virions and preferably bacteriophages as a control in immunoprecipitation reactions, where an epitope peptide is expressed on the surface of the nanoparticles, viruses, virions, which is recognized by a recognition molecule in the immunoprecipitation reactions. The nanoparticle carries a nucleic acid containing the sequence encoding the epitope. The immunoprecipitation is preferably chromatin immunoprecipitation (ChIP).

CONTROL SYSTEM IMMUNORECEPTIVE VfZSGATAK'OOZ

FIELD OF THE INVENTION

The subject of the invention is the use of nanoparticles, viruses and, preferably, bacteriophages as controls _in immunoprecipitation reactions, which are fluorescent particles. On the surface of viruses and virions, an epitope peptide is placed for labeling, and a recognition molecule recognizes it in immunoprecipitation reactions. At srmmnptemp!th,'iO dó v txe sk >nts í '!' ép p í<\ ni bibi BACKGROUND OF THE INVENTION .Ipötiuttpieiíipation tik and its aitalogies co-jimpunpreeipttácsó, CÖ4P) is a widespread method for the purification of specific proteins (or CO-IPbett for their associated molecule fekktsi Idrlénu «gyüfíe purification) from complex samples, including sej-Uzam-nekat or rapid weaving extras temek a; The traditional ÍF pivtókóKeK sep.hzs—A \<v s.óveti'en.ngenn.k\óxaí xCxdoduek. In the binding step, protein A or O is conjugated to protein A or O with dissolved antibody complex forms if» I /i knife of the complex oteei.Wu'a. ia ócninlxgáió'-'U'l, or náp'X'Cs stake fish? Λ' anogvo dökteVás is carried out with a typical western blot using marker-specific antibodies, or with a first-specific antibody, or with a marker-specific secondary antibody (or by detecting the enzyme-linked content of the specific protein). Any iu-iiiesi-based detection method, for example ELIbA. can be used.

Croinatin ífíunfufpreelpbitefei ICblfe) of the IP is a spmÁltku» area of application, which is suitable for studying the interactions that carry wheat-herje-T.)NS millet transplants, typically icbérje-ONS in, .The method is one of the most important «ΤΑ ibe-keso-u! » crnóu'Azi modvzci, amc.ynek has a n'k-oto effect on the Taxation of the cmgenetf\.o science o.- area.

We use ChiF-ixn np«kus,ut su'hzatumoi The mmtu et-xtlt-taso z-star Chromattn originating from the cells is relatively small feakcléii we extract the cronuain DNA-binding in the form of chromatm associated protein. The sample is , ~en vi»>b' et e \ ' s, , _f tk« o ^r e í g Í '-scl ) \aln >· V 'a'í' ik >> ni I, \ ·· . salt , '< 5v ,,-<ox κ \ í xí i í íven protein nucicln&av DNA or RNA interaction can be measured on the same principle

A ,χ , Ü-, h'pes xoar fxuldu- átV’Cvehet h»s«K nt\ ere sex a nu\>e nvok>\>' eke t. xpeuváis epitopi®il is recognized. Antibodies typically bind to beads, such as protein A, protein B, or protein A/B coated beads. With this method, it is possible to purify such presipkáí complexes that associate specific nocicinsiiv fragments primarily with feHsoteri protein by the aniüest! They contain DNA solar systems. Finally, the end product of the protocol is flour ltod leucleic acid. The regions to which the nucleic acid-binding proteins were bound can be identified, for example crownsafia-associated proteins, which are directly bound to chromaffin. thereby identifying these regions,

CbiF-hen typically uses cell lysates as a model, in which the rurkfeic acid protein and protein-protein complexes form buffers. mim fexáióxxerrei spit crosswise. As a result of cross-linking, the ricinic-iehenv complexes are formed together. to thereby obtain smaller Sragnicus that can be analyzed. In another version, krotnatmt rukutázekku! esne'-nut i-i'öeu in avu-tocat, and looking for-Ncs-, íczscuA aiialaban tT|F)

IŐ929ILÍŐI4Ö/SÖ

For tzoialf DNA, iron. other nucleic acid tests should be suitable for later tests, such as PCR, or QIV R basic tests, or genetic DNA sequencing. For alternatives; protein-based deicing methods can also be used, for example! it's WEIRD.

These methods also have a high rate of success. such stencils <·;; lack of controls that affect the reliability of the product. This leads to significant morbidity, which makes it difficult to implement the range in clinical gysskoriatha. According to the engineer, the following coritrolls were typically used. my former

Ai Negassv controls. nsins, for example;) cell physates containing no antigen, non-bmnnogenic, or uiáifierests aattitse preetpstation

B) Positive controls. what, for example, is sj antigen! containing bzáturn. or htMnogmdzátumpi) purified antigen ni) amigeni. soi ezpreasing transaene cells ν' Kdbt bi'xd "Ch vg\?'ko>t!O öke!'V ,1 kel;·',' nek this, >) cu a nn\is<. ,>t<e.'

I) IP flow for the analysis of the binding efficiency: is the first step to verify the precision of the measurement and is the first step in determining the overall efficiency.

About my positive stone! it can be, for example, aiikvot originating from brain chromalfii, which the antibody! step, or before pre-skiing, we don't have ·ονρ',ί· account!! Chromium is applied after kv-suztkoíé, kerevtkouNekeí is eliminated and isolated with QNS (or stucco acid). If we have PCR-based antlysis, it's even better! the corresponding PCR product is effective. The input data was filtered from mfofa, so that it can also be used as a positive control, we can also reduce the data normalization (see

Ntsrlng M. et al., Chrmnatin imnmnoprecipisatton optimlzatloo, qo&otitalive szssnlysíe and data norma lixatlon Plans Metbods 200?, 2 s I.). .Su-m unfnnnpsesipitalí chromaiin knnüoü (innal kosurol!) recommenders reference control. ntinsi in the case of both PCR and DNA cross-channels. As an additional positive control; does such a cutie come into the equation? which is able to consistently bind antibodies against ifeser ok fosom ov market in different cellular conditions. What about the IB? IDsKáutec ffometsiah 5 y»b and I s?d fosson !!)). or something like that. which are physisocated to svat'· átis regions (must í-ET-KOAe, aceíiláh Lysb, or ID are often assneiahed by the sMACmiflyTM Chromattn immunopsecipnatior· Restem Catalwg no 49-202Ί. 2? Jattusry 2011; Mcfcwest KR and ferguson-Smtíh AC "öistingusbmg epigenette smsrks of developmesstai and Imprinfosg regtsiaisosr', Epigestetie» «& Cbroirnttin 2010,1:2).

A negative control can be a chromain sample, to which we add a non-specific coníToHsmemf specific ímtíves correct' (nö-antibnöy. or NoAh chemistry, see! l-laríng. M. et al. 2ÖÖ7). Negative control samples are handled in the same way as CstiP samples. The NoAb Uhistákbo! the resulting QPCR signal indicates its background signals, which is the result of the chromatin analysis and the CbíP protocol?.. In the case of NoAb samples, the washing step should theoretically remove all the chromatin-specifically dense chromatin and thus it should be a signal; adnia, t cv m uok mcgko\ Inc·, .-um not >p<-fofktó ,f ^! üste.ue.\e!, ntm pek-fem nyal igu and mouse foC amdesses are spun as a negative control for the variation of the non-specific coagulant. However, researchers also know of an epitope hcmutnítnístel; containing particles and the nucleic acid encoding it, which is used for listinfinprecJpilácI methods.

.THE SHORT LUMP OF THE TALÁL.MÁNV

Imtntssprecipiráeiós rcakcisfe bau konnuli b.ztosnas^ta applies to the application of the felabtáfiy virus., or a suitable vsriönnnk. we are dealing with this ichsroefo molecule, above all on the vson. or « sinvn's gene pool contains an oligonuclcotide part, which is presented by the virions and recognized by the recognition molecule in the sx mtmuuprecipitation reaction.

According to the invention, the uanotoparticle is preferably a virion,

The invention is serval. the oligonucleotide segment is preferred to a traceable oligonucleotide segment or region or section.

According to the invention, the oligonucleotide present in the microparticles or in the viral genome preferably encodes the epitope. Lg) according to the first embodiment, the virus or virion is a bacteriophage.

The invention originated from the ^? mmtunprecipitation reaction advantage;! oncletrisic acid immunoprecipitation, the advantage of which is direct nacletftic acid immunoprecipitation, or complex immunoprecipitation of ukuleic acid, the advantage of which is chromatm Immanprecipitation,

The subject of the invention is also an immunoprecipitation kit ricagen kit, which contains · the molecule that recognizes the antigen for immunoprecipitation

- a positive control sample, which contains nucleic acid and an epitope-carrying polypeptide, in which the nucleic acid contains a nucleic acid part that can preferably be traced to the cs \ag\ code, this epttop pcp'idvt

In an alternative arrangement, the kit contains a recognition molecule that recognizes the epitope of the antigen during immunoprecipitation and, as a positive control, a gene encoding an epitope peptide in the genome of a virus or virion, preferably a bacteriophage, which is presented by the bacteriophage and The known trees of the generating molecule during the immmtpreei.oiiaeiation reaction. Preferably, the copper sulfate is part of the precipitating kit or together with base phosphate, preferably with ChlP. According to the invention, the application of the putty is mentioned as part of the solution, and it is considered to belong to the subject of the invention. Where appropriate, according to the invention, it contains;

negative control particles, or a virus and/or virus, preferably a bacteriophage, which lacks the oligonucleotide and/or epitope, but preferably contains a nucleic acid and a polypeptide. which preferably does not bind directly to the recognition molecule.

tools for the recognition molecule. to detect the binding of antigen to the sample, preferably the positive control. year may be given; negative control, a carrier for the binding of the detection molecule, preferably a bead, a tracer reagent.

The kit preferably includes tools for antplirikasrio. oligonucleotide primers as DNA initiators, preferably:

. rt R p .ku! t bet t iu v ¹ ·. ·. tm,>k,->k<lo vAv«ua <« ;'( k, >tó;ft' with pECR primers for the bacteriophage sequence ampirt cell.

The kn érOnyó<en tarta unsz Ζ\?κο.'Άδί the ouMcmsav, \, ₅ ev au female eg> part of H'kvenaktsahot to which the unrt gene, or its calbebet is attached

The kit may include a tray with containers. Through - can contain the kit element. A sample container is used for the analysis of the tested sample, a control pen is used for control measurements.

According to a certain embodiment, the sample container and the comroll container can be the same. The invention also relates to an imm-Mtptvtupitation method, which method includes the following steps:

provision of a group of samples, i.e. several samples, or at least one sample, which contains antigen or epitope-carrying serum, the addition of the antigen's antibody, epitope-recognition panel, is performed by one or more agents, the epitope-carrying peptide and the nucleic acid containing nanoparticles as a positive control nanoparticle, in which case the nucleic acid contains an oligonucleotide segment that encodes the epitope, or a positive control virus or bacteria, for one or more samples. additions, in which a positive ktmiroll virus or virus contains encoded in its genome the oligonucleotide sequence encoding the epileptic peptide, in which the epileptic peptide is presented by the virus or virus and is recognized by the recognition molecule in the immune response addition of one or more samples to the negative knnlroll nasuzikii, virus, or vlrion preferably containing ollgosakleotide aeta bakleriphage is the control, or any contact of the controls with the recognition molecule or contact is possible? tea let $ !,'?, ,-í «witness g ieetpr,u n·- leakető ke, dók,mset, the detection of the binding of the recognition molecule to the displayed epitope.

The positive cone is intended for nanoparticles, viruses, or virions, and those that are used for the recognition tool, can be advantageously compared and can be considered as indicators from the point of view of the immunoprecipitation reaction, where the higher number of those bound to the surface selected means a better performance, i.e. a better quality of the immunoprecipitation reaction?.; . According to an advantageous embodiment, as part of the detection step, the nanoparticles bound to the recognition molecule are those of viruses or virions, and the number of recovered particles, viruses, or virions is compared to that previously added.

According to a preferred neutralization method, the nucleic acid immunoprecipitation method further includes the nucleic acid modification and/or the binding of the antigen and the Iragions bound by the antigen to the recognition molecule. in which the recognition molecule is preferably bound to a carrier.,

Fragnation of Λ -uAí einic acid is advantageously carried out by sonication and/or nucleic acid nucleases, preferably muscimuscular nucleases. we carry out

In particular, it is an immunoprecipitating nucleic acid inhibitor, its advantages are chromatis immunoprecipitates, DNA immunoprecipitates, and RNA immunoprecipitators.

According to a preferred implementation trio: the bound or recovered nanoparticles, viruses or viruses are analyzed. It is preferably analyzed by qPCR, sequencing, preferably detection as a fragment. Preferably, the detection of the recognition molecule attached to the presented epitope is. we carry out the following! by amplifying the presented epitope coding region by adding fragments from a particle, virus, viral, or preferably viral and/or antigen-bound micelles to an array containing several nucleic acid sequences that preferably provide at least one complement to the nucleic acid sequence that belongs to the epitope encodes it and/or by sequencing the nucleic acid bound to the antigen, and using the Brain immunoassay method, e.g. with an immunosorbent assay, e.g. EUSA.

The recognition molecule is present in a cell bound to a carrier at the beginning of life. In appropriate cases, the recognition molecule bound to the carrier can occur before, after, or simultaneously in the same step as the binding of the xniero molecule to the antigen or protein.

My lek-m 7Ό n\í\kv,'a,n\ the uoogen cntOpm ' s<o, ccdeheiulK. 'órtetó Lmot. m s/rmn dv'íestátim l'te <\cx'h ' h h-nete p > ?\ lutuk , ve f.mupnok < iwu ,ό-ί, >\> k,\<\ ex nv\eg>vrh i r,' N for Mars at the f.«gs.i súm-x ϊ »η·ιι\,·« Az, smis, v,o.o '.· !κ·!ί ,w»nhA gv'no'mu\b .í.'t .ız epipnp or contain the "nmufsogét part" of a micleifmv protein, in which the epitopeptd or the immunogenic part is presented by the vmon Gertii, in which the peptide or immunogenic part is ?-2ö preferably 8-18 amino acids long, cut- 5 - 22 amino acids preferably at least 5, 6, 7, or 8 and at most 10, 11, 12, 13, 14, 16, 18, or 20 amino acids in length: and in which the epitope peptide is capable of binding to an hc-redczo: bound recognition molecule, preferably protein A or pasiéin G. or a combination of these.

According to a preferred embodiment, the viruses or virions are essentially the same, except for the epitope or the Immunogenic part and the nucleic acid sequence encoding it, and they differ in the epitopes or the nucleic acid sequence encoding them.

Nanoslots, virtssoks, or v trios preferably form libraries. Advantageously, the library can be enriched in several enrichment steps, in which there are molecules that specifically bind proteins or homologous parts. DEFINITIONS ííi;,'?i.víipf'e<?í'póíh'í-f fi'F;· case is a method that involves antigen binding of an antigen, such as a protein antigen present in a solution, which uses one or more recognition molecules , which can be specifically bound or bound to a given cell, in which the recognition molecule is bound to a solid S'asic substance at a specific step of its processes, mkiúai no. its transmitters form a protein complex with the recognition molecule and Thus, the sylacid phase suhsiratho? binds the reaction sufficiently. This complex then transforms the reaction into the primary _step or event. The process can be used to identify a specific protein. for the purpose of tómc-nvitation Λ recognition molecule preferably antibody, cut any of its mszl-zte utv, c.nuuxa ;Vaáö';>.vms'ovfawnp'cogvtnoec Such a preclptfáclós modspr. in which in the jmmtmpreetpnauví· reaction following immunoptecspitation it is. the complex of antigen and the recognition molecule contains nrskieinic acid. In other words, ko/t put away \ag> indirectly and nucleic acid binds to the smaller molecule·. The direct maternal acid is a nucleic acid in Imsopmcipiiation. antigen and the recognition molecule, for example an amnesty, itself is known by the tmkleInaavuí MA oekkm-,,η-protein complex pmedipatbmt the recognition molecule recognizes a protein target It recognizes and binds to it, and it is bound to a specific oaklemic acid, for example it can be DNA, RNA or other double-stranded or single-stranded nucleic acid <ds or ss>.

A>oí>;cíííu pífí»;«g?rí?í'íoídcíd fC'ó/F); It is a specific application of IP and a change in nucleic acid immunoprecipitation, which is suitable for the study of interactions between proteins, proteins, and proteins. More broadly, ChlP can be used to determine whether a protein interacts with a designated target protein. in which the complex of the recognition molecule and the precursor protein contains sienna. micelleic acid, usually tachamic acid aagmamil bound by the target protein. In ChSP, typically a long nucleic acid, which is either DNA, or RNA, or genomic nucleic acid, typically chromatin, is digested and these fragments are used, and that fragment is obtained by ChIP. to which the protein binds. The product of the IP reaction is typically a bead, preferably a bead coated with protein G, protein A or protein AcG.

Www ííío/cóv/íí: case·· a macromolecule that can bind specifically to the target, which is a polypeptide, or·· a protein in which there is at least one·· antigen recognition site, the recognition molecule is variable ί>

in his region. The variable region of the recognition molecule is a region that can recognize a molecular surface in the native region of the recognition molecule that can bind to the target. In most of the recognition molecules, the variation of the variable region is significantly higher than that of other parts of the recognition molecule, the sequence identity for these places is typically low. The recognition molecule is primarily a protein and its variable region essentially contains amino acids.

V >>,'<-<·>,Vx> _t >i<>>p>( ,ü\\ uwi , · t xm d\t> n ' vt ,md\ -gx.. sisu- tn can bind to the target, at least one antigen-recognition site is found, which is present in the immunoglobulin molecule. It is applied to <fek, but also to their variants or fragments derived from them (such as Fah, Main F(ab'2}, Fv) single-stranded (EcFy), mtmohody or mutant fusion proteins, which tamlswz a part of this antibody, or other modified configuration of the Immunoglobulin molecule, which contains: an asstigéu recognition site, or some artificial version thereof. The antibody can belong to any class, such as x dvi <·<<' \ r sgv C'M ís m \tm« _; osja» t <>., with a distinct, well-defined class. The subunit structures and three-phase characteristics of the different classes of immunoglobulins are well known in the literature.

&;ι.·Γ \z epssop this o.tan heh on the eetpout. ome-h edpom poítpepod, tag> cow can be a molecule, dunsós<n .nm-k n< Ll.u»u - 'd- xn uhlv te* en ve-e ; t. au u stet<mx,l· voto ej. » -ge<sí sheep; x'UK ⁴ according to the invention, the epitope is preferably a 5-22 amino acid long linear epitope, which is preferably S, ö, ?, sagt k, é'desdeh.dh K>. II, CH 'd, to, ík.vage ?0 am washing ut 'ut talna?

V ep top .'>'?!! es.\ -'hun heh u celnxx, kuk!·! nmei'. poh.vpnd, vagv fekry'nok'kclu can be located on its molecular surface, to which?, preferably the antibody or its binding region is bound with the same specificity, mmt a? c»!k>phi>? You can make mime balls!; for example by selecting from pepid libraries, preferably bemums» phsphp. t nx>dszsr, or protein engloeerlng methods, such as using in sslieo molecular design, The mimofop in the description» a certain head epitope has been superimposed,

In the application, a particle typically means a particle with one or more dimensions of 700 sas or less, Söö nst or less, sofi mn or less, 200 nm or less, or 100 nm or less and the so-called nanoparticle is a rmkleic acid and a cphop or contains mimotope-carrying polypeptides, essentially contains it, or consists of it and, where appropriate, its bortioz, or its surface. prpod \,'t,x*h,t sagt butokfe^enzbzz «horse-tsíW'', d„ not k«»ard»g<,\an préipeplsa or·' at least one part of the protein is coded by onkfem acid, which part contains the epúőpot. According to an old method of implementation, the nanoparticle is shouzo or ose.sterstégé. An old implementation method is a seriöt & oaooparticle virus or vlritsn,

Accusation in the petition? We mean an infectious agent particle that is capable of living cell replication of an organism and that particle essentially contains a nucleic acid bounded by a protein envelope, or from it, in which at least part of the pre-envelope is encoded in the form of nucleic acid. Virions are capable of infecting any organism, from animals and plants to fungi, bacteria and viruses. Good luck at the castle! transition particle.

In the submission, we mean a particle of an infectious agent that can infect itself in a living organism's cell, or can infect itself in the same cell! It has a nukietnsaw&l (or its helpers), which can be found either by itself or in another cell in the same cell, which encodes the protein of the virion.

The ooteríö/ug is a virus, or preferably a virion, that infects bacteria by injecting 8 genetic files into a part enclosed in an outer protein coat (capsule)?? is located. The genetic stock of a bacteriophage can be ssRNA, d--XNS, ssDNb, or dsONS (where Ds-' or' 'ds-' prefixes mean single or double-stranded} in a circular or linear arrangement.

The epvop or popsid is immobilized on the surface of a molecule if it is present in a form suitable for recognition and binding by the chemical, or it is attached to the surface of a molecule. The recognition of the target by the recognition molecule, or preferably specific recognition, is an event or sequence of events when the target comes into contact with the recognition molecule and the strength of the interaction between them is stronger than the recognition molecule and at least one - but preferably more than one - different transfer between destinations or transfer/transfer under the same or similar conditions.

Is the AoHhvV of the IP or ChSF posölv a control substance or agent that, when added to the reaction unit, has a value that increases the signal in the target side? regarding its existence, or regarding the occurrence of a ttnmunprectpttactic event. The input control is the l'P method, preferably a positive control of the ChfP reaction that contains a wide range of target molecules. which is primarily the execution of the immunoprecipitation step?: we win and is suitable for detecting or analyzing the target. In the ChlP reaction, an e-absorbed nucleic acid, such as cfomatin, can be used and analyzed for the presence of a target sequence. complex can be detected.

Vi%i vn rcltvnt iCkmfete relkrb kontrol’ ewagv our fehs.tuto -women·}!.? n-feuk-to as a woman? ,u?v this rwi indicaifv & nei?t for specific recognitions and bindings in an IP or ChIP ntresult, The nucleic acid found in the control either n&noparticle, virus, or vliortbas? ayomo" is traceable, and in the submission, do we mean that its presence can be detected, or is it a monitorable, primarily quantitative" measureable base axis? töötisenel, your samples are detectable and preferably the level can be measured with PCR, among other things, 'P v h_ ic ancient ti\vertAid ti to ν-,ο e o. r s~ using methods or a combination of them. About my stone! function basically requires what of the added control quantity? what fraction of the quantity is recovered after the IP reaction. In a preferred embodiment, the imprintable nucleic acid encodes the epitope tag.

DETAILED DESCRIPTION OF THE FIGURES

1, Ocher; tid.ai hUOvkon.Uf- mmoangeo eettóp pepf.d (Pt? kodtóv Oeks.nc',,'. e\ Pé-Mtóki- tag '<<-Dm koostract A· ü&nsiáeion of the sequence encoding Pá pepiide specified in the text. The. mi.sRE-Pl.· sequence of phage DNA construction patv'lálls nucleolide and coded prolein. Abbreviations; M13 phage gene Hl start codon , >b. í lo\ _v <.0,s tz>al'wk^ttvía Vvom « tgl ?cAs wo-etde ,1 .tz hN ?\ Uett^ irt eggceg, light gray), P4 pepid coding sequence (dark gray) phage germ Ili protet?? codes? sequence

Figure 2; Pa-M I.IKf presenting the ΡΊ pepiide: broad schematic structure, A recomhináns fiiamén! Mi lago?

E.eoli C.R272S was prepared in the Μ13KB flat-stranded vector electroporation, which contained t? Nucleolide sequence encoding P4 peptide, which was cloned to;? The protein of gene 3 (its envelope surface) was lysed at the N terminus, and it is assembled in phages and cells. & from the co-constructed positive strand of the vector (single-stranded form, «.DNA, 2722 bases long) and the bitfokfebebs, which contain the ?s;njo{ gét-b protein (~3?tl0 köpúO, ίΐϊίϊϊΟϊ· gést ö. gene 7, gétt IX é> a P4 , and Π s. married te e t f S kópsa). Recotsbinans P4-M ; K,;· Kg o/csten i P4 pepodet n tcís/re.en jCeiUh η» > es ” osj ;n, C-. switched to ' kodo\> OXS-sei 3 átn<S M '\t '' ΗΓ'Ηί'.Λ,'ρ 4 ssAhSCf^I 3.

A) Sequence of antisense fibers for ΡΊ 2 SM b and P4 11-M11KC kions, the M13-% sequencing prime .3ik<Uiuara,-,3vsl ik» i ngbnd Htniafe) rag i fCGU f k'U akthuAaV dl-áo -vsztcso , ΪΜ peptide reading v.to. i ^L< B pvi<> _s xne?s.ir\e KiOn I le , Ars IV <v<hg s kei B ' ^s g' Sí H-,e sC ΓΙ start coóoo K2AC on the antisense strand, middle gray 187Ί í? in position

8) ABÍ 1 '0 .Inaugural DMA sequences prepared 04 2 M1 1KE kló;! sequence. The P4 M 1.3 lag the tsegativ !aíbsszenoo vta' tzekver,sást -K' sugarsalopete to the'kaiota.'avavávu' would sneak Eagfelv ttVAl»'Ctí> al ke position W pepiid reading seeker 88-115 position ACC ball place (ΟϋΓ .At .'C.i 52-57, Mii Ki?gets Ili start cetiott iCAQ: 187-189

Figure 4: P4 specific QRCR essay used in the screening.

A. Schematic of the arrangement

8. Chair engravings and melting points

Figure 5: Use for the Chik reaction! chromatin fragtneni: meter attniízise Agiiesd Iliottnslyzer-reó ó A. shra A f hií ^! Kg >p ke 'Mseslcsev atnpofUvtos curves million, HSOezeres 10,000 thousand HDAC 1 specific l'-í members llilACl atitiltcstíeS gytíitötök S 8, sióm: branch spike experiments after ChlF reaction tsífio, Kklezcr and 10,000 thousand HDACf specific P4 Kgot BDÁC5 collected with antibody. Figure 68 is the ó .A. Figure is a technical rephkättsma b €. Figure: Amplification curves of spike experiments without ChIP reaction. They are isolated from the quantitative input of the intnnmprecific step containing DKS i milito, Kló ezer and Ki ezer&goí without the reaction.

6. Bábra ChIP •cakoíó'tetküh tár <oAok set.okA .Bttohf k.ase- eötbet 1 DNAt ί>η ^; '\·, t»Space from 1Ö% amount of input of hmmmpreciphation Mxó nélkhii reaction containing this phage (wells 127, Eh, f9) and i soolli'5 kroov.uaunenies dilution (1·? 1'8 l'4 .and holes) were isolated. We have carried out immotmree.ipitaet re.-m, oh Figure K: t.'hlf reactions asnpü6káck« curves of broad spike experiments. The i>NS-i ttDACf specific antibody was isolated from the Uutaimazö CidP reaction and it is a l'M iag particle. represented by the following negative control reactions: empty branches of 1 million, 10,000 and 10,000 copies of test-splicing MidKE.

é Figure F: Antipuffification curve of broad spike experiments following the ChfR reaction. ÖNS-r HDACi was isolated from a ChIP reaction containing a specific antibody and I million, 1 tXi thousand and íii thousand R broad particles are shown together. r<.,t\ <of, anteb tntn rvo.vzC'ít the epitope sequences of the Ili c\C1 Specut kas antibody ah&i leiissoett, ó Figure G: Cp values of Spike QPCR reactions. The QPCR reactions were performed using the average Cp ,'S at.ag s\u icon <h's ctGok:' in the figure. tnut.intnk 1 .'Olbo pOí tttv kontsoll Kg the JítöKus basic theory A1, A2 As pataiéi oult a^-s^'pv 'κΊΟ-ί-Ά st 'A,?', v V Λ ^r <'>k \\ ht st >et< ká„ t' o iirtpiikáiutnokháii. 100,000 members for exams. 7 ieehaikai replifenb&ts and 1 QPC8. measurement takes place in Bt, B2, winter. and B4,115, Bö lynkakbats. For example, we measured Kt thousand phages in the Cl,. C2,0, or C-4, C5, €6 Ινοκάκαβο,. The input represents the number of units used in this preeipitation boz,

Figure 7:

á t&g\s< v.tá . k -V, í„í os terelsz,nn K se ^f > n ns^Ker tkKU'reered'oefSí. n. C' vere, _t p message sequence data for the first 100 analyzed sequences from the 2nd enrichment step with the MIICR2 antibody. 3. Enrichment for the MECP2 antibody (conformity, specificity and consensus). step by step for the first 50 analyzed sequences. We analyzed the data with tnintox software.

C λ kums.nak t kér dux u- műm O ’P uw/atneséMí t smího Og\n -«ΖχΟϊηοζ moutotrgvancey cherstok back and raénUk te QPCR relative anti ttkáesós method. Results from rounds 2 and 3 of enrichment with .MECE2 and p300 antibodies are shown.

Figure 8:

The results of the regular proteosequencing of 154 juniors in the libraries Kortzetválkság. quality and score data for the top 100 analyzed sequences from the 2 Enrichment steps of the MECl'2 amliesn.

R kt taxtg, quality help. hom.'et »,i>data u W ClD edttestk í töttes.o * Dusánt .CpőKd sz-Orsaí.. first löt) analyzed for sequence. Data to raimux software:! we will hire you.

<2. Ck!P vinegar comb of libraries after 2 rounds of enrichment. Input from 1 million Tisgúx was recently recovered and tested using the relatively anti-inflammatory method of QPCR. The results from the 2 and 3 enrichment diseases are iou-ataik ΜΗ P' o, $. itat rr.úsxtok-tC

Figure 9: The mimotope quality indicators and the tree. recover libraries ChíP.

A. The quality indicators of the unique poslcióists of the snaiizsbt libraries for Mimox software? we counted.

B. Summarizing Mitnox's quality matatees of several different antibody-stained libraries and rounds 2 and 3 of selection.

Flat diagram: Attenoic acid concentration indicators after 3 rounds of member library selection with MH2P2 and p3öí) antibody '? Figure L \P i mg 11 -S \ ctrenw^gt my letter I tml.m taros konts ass sres.mjsstas gventuszn my article which is a concentration that is much higher than the optimal concentration of spike control.

Figure 12: Mapping beneficial modifications to ChiP'Secs. The H2K2? The biston acetsiáeio was mapped in technical replicates. It shows the region of the ADR mechanism of the mouse. figure. The CblP-sec analysis was performed on the üintnina sequencer.

Figure 13: Non-complete, trag'métaé só' aram hagmcnt n e-et eios/lus, II $ & -somksssot motto nukieoszonáiis fragment touriig ^egerrnk a big gardje complex'!·. szeie^hemetnok am<l> je'vü'ósen -.shockkeuif the transknpes lactors ChIP 'naiekonysngm.

Figure 14: The chromatite; ONase-based text editing

A. i-ragmení measure Agilent Biomsabzer size market fragtnent size distribution ie. Agslent BiOsnnlyzer results on gel-like reprazenıaclő Ixorintaton ifsgnvent measured distribution. .The first column « panel 8-han reported', he marksemket shows the 2 Dxzlnp the miioocncealN nukleaz etna; .-10.- núlktll Doh!; His DNA mat, the 3'9. Columns show triiephocoeeals obtained by nuclease digestion. DNA in a sz-ms ratio. Incubation for 15 minutes 3? We finished on C.

DETAILED DESCRIPTION OF THE INVENTION

Can the inventors come up with a method that can be used to make spike controls that have similar properties to krotnatite? examines with the immunological method; protected-DNA complex, The method is based on the use of an epiphobe presentation system, mini kosttroii. The epitope presenting system can be used as a positive control, atstetmytlxtí? contains a specific epitope, or as a negative control if it does not. Can the eptope presenting system be a variety of oarsophets that have one? with a polt peptide containing a nucleic acid that encodes the epitope. The epitope presentation system is preferably a virus that encodes and contains the epitope, preferably a bacteriophage.

In order to understand the functioning of an immunoprecipitation test, it is important to have positive and negative controls for the test. The laboratory must indicate that it is a suitable positive control! for a co-worker to determine whether the subject is working well from a technical point of view. The negative control defines the background of the experiment. The difference between the signal signals measured in the positive control assay and the negative control assay characterizes the dynamic process of the reaction,

A significant advantage of the controls detailed in the invention is that they contain both the appropriate target epitope and other mtsk'-us.oi; in nmeks the ntiklosnsat ntootoe can be followed e.'áhJ cet mtmm'PrtóiprnL ms shooters of the lakd máayknntroí! microparticle for the recognition molecule is stone-Old. as, for example, a body and preeipitaío', in theory, a bed, just like the food protein, or antigen itself. k.zerf, for example, works the same way as chromaffin taaga in a ChIP experiment. property of é.za. foiha.snálhaló on several levels of quality assurance, including himself ,ν í* 0. t\< u on their behalf , woman peio.i.'i ^!Í 'd ' egt rtup et 'tA'l'P vo u v.'tp beer s dl -^mpt ;> veins of Ch'.P words ás (Ch IR- sec}.

According to a preferred embodiment, the nucleic acid contains the sequences encoding the epitope, and therefore the epitope encoding sequence itself provides opportunities for control! for detection or monitoring, i.e. in this case a positive control; from negative Coptic! in the case of its itiartya we detect or track it, Vn-ülts system ,;lg.dm.;zas,;v J ertosmm tooth system did the inventors develop into the arc? a positive control for iofeamattn, which is suitable for monitoring the accuracy of the method. On its broad surface, it carries an epitope that binds to the recognition molecule, preferably the same peptide. which was used to produce the antibody, or its decoction.

As a preferred example, broad display methods are used to provide controls for the invention. Pepioid phage display technology uses expressed peptides. in fusion with vital proteins, thereby the peptides \riti, 5 tgo \ su ,, ¹ \t „ v\p ifw '0 0 1' -rtrt·. v ί x --et ati gönc » m alot u”t\ \ n fén packaged as single-stranded DNA found in phage, this. enables the identification of peptides by sequencing the corresponding coding region downstream. In ktomafin ptecipteation experiments, a recombinant phage that displays the epitope of the monocyonic antibody makes this possible! shirts checked; use. In the case of polar antibodies, a phage mixture can be used to increase the efficiency of the antibody-inducing reaction.

\μ„ίυο\.ι’ΐ! the* ? -λ t m rt ' sec. oet tticrt atuedc e 0' ,rfe tnox bt^rtt» ;>he, \ s fafe ? She ? a > ntn the po/uo control can be used as a spy control, because the hrttrtpopresuppíáing reaction can be added to one of the hrtttpopprepíáing reaction, and the first line of the hntnunpmdpiiáclé can be done, thereby properly characterizing the reaction. For example! the mgm can be added to the Momaho ton as a spike and its level will be símmonpreclpd the ChíP reaction ntan tss.mtms HA$ ben uiomivte/hahuh bto busonloan oanmiysm row», anmo epnop p!öentáhfe,tvt and able to encode the genomes of the same epitope , can be used as a positive control 1 u present finder attbats.

As a negative control, it can be widely used nmh »n>s,<4oh,* au,vgult oepnde, ⁵ ra spike of fsgtutk the non-mo'thvns kvK\Uy?' ηρ, η. '.mmmrutr a NLrug es _i fe'zo\ ^k 'ma gsornckmr· \,«rv t tonamafe nt latnad for antibodies, ngt «Ή.1 a 'mmavag \<x> Ό QR' kt^w dh,!'r' ,t/av.o.nl ametj -pemftkus & pemkíet iam, or the empty Tág, we can simply call the dynamic range of the measurement. The QRCK specific to the feg detergent is a quart; provides native data on the enrichment of broad-based immune-predicted spikes,

Based on the invention, a plowing optiop presentation system can be used.

The strawberry-presenting agent is a nanoparticle that contains, essentially contains, or consists of a mimic acid and a polypeptide, or a protein, or a protein containing an epitope, in which at least a part of the polypeptide or the protein contains the epitope-containing nucleic acid. IszalPsi is the epitope of the nucleic acid color; encodes, The particles produced in the literature can be used to determine acid and a bile acid, or a ün. carry an appletop with a teaching pohpepb. For example, an ipposaoma may contain ttucleic acid: while the polypeptide containing epspept can be covalently bound to the membrane, or a mesnbran ho.moto svgstsegevev psl-'-td menmmr p-sm-mm- pohpspttuksnt Alurtnov tm\hm msttd ,t nuhfemw, untai a pohpeptsd a nanoaraoy can be attached to a particle. The natto particles used in the present invention were produced by Seoít S.k. (Nanoteelmology för the blogger, Journal ot i.eokocyiz Biology Volutne ?§, Septemher 2.005. SkS594j made public. Mthietic acid is present in a gelatin nanoreizeeske capsule, e.g. poilcationbat;, while the target pobpepítd can be attached to its surface. In the solution according to the ialakkány, aihabaazkstö is advantageous nsnoreszeeskék e.g. those of you; published on 3rd of July 2008, US 2Ö0S/01 díXfeő Ai No. US.A publication document discloses jí-ferbeiy .1. et ah. 20081

According to the preferred embodiment, a virus or virus is suitable: according to the invention, a viral image; for example, according to the invention; oanoresese vlrns or vlrion The vlrns can be chosen from the following and belong to any order:

C&ndovtraies, Berpesvtrales, Monooegavirales. hhdovimtes, Picmnavtralea, Tymoviraies ami fesgameavirafes. Ao vu^ >H)t 4 e. ^{( _} ki tt <eo Teád the s.- RAS se eaters can be ,-cucv m >, or^y,ηη süss s»k.,The virus lehei hneans, or mrhulans gemmsp: l-gs preterah elrensiezvfehen a uras l'JRS mrus , penfenl dsuJAo vlrns, in the case of kiumatm psectpttáel, your examples are adeno cry, or adeno assoeuth controversial, s-brain RAS virus, for example tetro virus, RAS hmmprecipitaeíóí. Viruses especially the treatment of adeno viruses is íSiermti eg, the stone-like manual: ^ik Virus Conslrucrion felt - The Mannaí Rreiburg Btoware IGÉM 2010 (Available e.g. at the Freíbourg Btoware group and on its website}.

In a more preferred embodiment of the invention, the virus or virion is a bacteriophage. We used the M13 phage system as an example of the system. .The R4 peptide cotiold nncleotide sequence used as hell is sbnbkaotiosi. why the hell not x? «ette n puffed gene Ili fenenéhce mfemo The terntsrrhs fetempt Vonhan a tag i nmeís , even tsAérpje of which vsat surface test, nlkalmns epitope tn;rtki:ms, _li , _í , .urtely applicable according to the invention. For the display of specific epitope peptides, or for the selection of specific control mixtures, or for the selection of monocytogenes, especially the gene g protein, gene Ö Heitja, and gene 6 and gene ? protein groupAd ·· patterning protein uses•χ·

I ' ^s 'éti I de < ». oops! \ ΚΆΙ fct * s Ϊϊί 3 nett M 'tee< i <. 'hb*n ΊΙηο s , <> s ?rp < - fit V ' vekRsr eiekiroperscöjávíd, The packaging in mature member s cells is assembled from the constnict positive thread of the vector and its ttnuktei í\í? \'i as \-5\ck a yen \ Bt k-ttenc í ? \»n copy\u es ,> m no' gene 5 t, Yit, 1A they could do es. the Ha sva Hí fusion protein (3-5 Kóptaj a reketnbioatts P4-M 13 thin construct is displayed on the surface of the P4 peptide and is physically connected to the DNA encoding it, Fig. O). Both Itltkns and llzogetic phages can be used in the current situation, since their ability is determined by the immunoprecipitation reaction, only by the propagator of the virus. Bacteriophages used in the invention can be, for example, but exclusively phages from the following families: Siphovíndae, Myoviridae. ítedovirsdae, Lipothrixvirtdste. Rttaiviridae,

Gystovlrtdae, Fttsefiovjt tdae, Gsebnksvirldae, Gnbuvisus, Cortteovirtdatő Ciavavisidae. BscaisdÍiYirid;te _; Daciiiovsridae, Anrpnliavirldae, Tectiviridae, Plasnsaviriiiae, Microvittdae, Levivlridse. inoviridae.

Based on the model, they can be the logos, but they are exclusively lasnbdst logos, "According to the model, the bacteriophage breathes, eg lansbda tag, {X phage.t, the Ϊ sticky Cg, e.g., the T7. phage, the Ί4 phage, the T7 phage, the TI2 broad, a KI? tag., an M type or an MS li'pnso tooth, e.g. Μ13 phage nose brain MS2 phage of the G type broad, mini eg. G4 fó,g, the P type broad, bake: pi. For example, the P2 hangs or R hangs, the Pisi tag, ssdíss eg, Piti X 174 ing, the Φ6 hangs or Φ29 wide, a N wide bake e.g. Na fág, 1 Hő fág, however, it is not limited to these.

ChlF klaerlct: Á CIslP experiment with a preferred method uses controls based on the aiablos \ : sliA'^k ¹ d 'aU'S oeited ·,, tkoewí '< os ,os cl , \ c/ó ck \ s however, pékía-sl the histone proteins or the sucleosome proteins are necessary for the synthesis of Inaísv ChlP.1 (Barskt A e al. idesosbállón o;'tratísersplion faetor target getses by ChIP display. Mctttods Mól Bio! 3004, 455: 177 170.1, In certain arrangements additional, or We can use matte cross-linkers, for example distskéúsúnidti gluíeráO in order to keep the protein-protein complexes The cross-linking reagents are the complexes,·.

The krornaon tor áifaláb&s; Nucleic acid s fragmentation is a defining splenic year. It is important to achieve consistent syntheses and reproducible results. The mogkiváut iritgsnoat tnéret is in its main ancestors between 150 and 501) bp. To the extent that the fragmentation method can be trusted, the number of fragments is increased and the following detection step is the analysis of a sequence up to 2IXMB0 base, then the fragment size of 101) bases is preferred.

The size of the ihsgssteat size is determined by the size of the meskiestoma, which can be affected by rsnkieítz»» organisms. It is a general rule that ^; In comparison, the iVssgsuesn tstestd, austal faster fragsrwmíáeio can be obtained with tssdilyase digestion, for example with ímcroeoccah nncleases. The fragment size required for the preparation of the iragtstens libraries that are required for this is from 1000 to 1000 bp, in this case, or in the case of native f.'sdP, the digestion fraction is preferred.

The next step is ínmmnpreespititeio, which is cruel from the eyes of the ghosts of the successful ChIP. In this step, you use a special reagent, such as smeester, to bind to the tested protein and coat the complex with a binding agent, such as protein A or protein G, to form the beads. to finalize the petition. The beads can be magnetic or Sephamse beads and can be collected by magnetic extraction or centrifugation. The pearls can be marked with tsitalmaztonafc and can be sorted & marking aids The formation of the complex can precede or follow the breaking of the fslissrierö snoieknla to the support.

This is due to the fragtnented krotnatsn ntissia düssia in those lyagtnents, asnelyek isrtaimnxzz&k to a specific place of the nakiein band, the white of the cerebrospinal cord is located in the frontal part of the ChIP experiment.

on the degree of enrichment of iragmens, which in turn depends on the quantity of the chelating molecule or aniiiesr), while many bodies are ChlF-recognised, the mttiíesíeker in practice are numerous in the area lus/nálpk And in the case of (biP validáh antijesO'S) this k-pests to the cdenor?c«e belt the corresponding control-ka!λ talahootn aims to solve this problem,

According to the invention, the positive spike control is added to the experiment before this immunoprecipitation and after it is recovered in a satisfactory proportion, while with a suitable negative control no or significantly less spikes are found, the immunoprecipitation step can be considered successful. So, if the target protein of the same or very similar epltop does not have the dttssdás, the problem does not exist in this step. A suitable negative control can be a non-specific control antibody such as ant; Verbs and/or a verb or phrase that contains the search ephopol (or íohnosópoO, »g> t tvi* -x p-onkt s'ketcjj>'gc alapretcvn iugg ,t tamdnsám u .rort febere salty spe<.Tkosanly tied Menüin, oh;,-,n\naro: onme-- "ot'tcoc» tmmsrC'uvn revaen- that roias, &λ-'λ0 ^rzi-'t?»'·- ,') n><-.va ONS öösoBís·! we reach a specific binding site, compared to a non-specific control antibody, mim péb . \l„/an <<x<a'td η mt esüctsi Bt.- Abát s con\ sok, c

In the immsmprueipjtáoio reaction of the landlady, it is. sample to be analyzed. i.e. the tested sample and the control minis are present in different tubes or containers, in this case the positive control or negative conlzoil of the control of the invention is added to the cootfolbniniaboz·, which is in separate rain from the sample that is being analyzed. in an alternative way, the control is added in the same container, which would contain it; given sample, in both cases the immunoprecipitation reaction does not occur in parallel, but simultaneously, in the same g-reaction, in the \x\galt minion and the control ion. In this arrangement, in addition to the control and tested sample, we also detect the reaction from the fly. This is possible with several detection methods, such as, but not exclusively, with QFCR-reL or ChlP-ehlp. In a typical case, an aliquot is taken from the reaction mixture after the reaction. If the detection gives the opportunity to follow the reaction and the control and tests! sample sign between you! for discrimination, such as in the case of QFCR with fluorescent markers, in a certain arrangement the value of the control and the value of the target can be measured simultaneously in a simple reaction, uz „s O ' tb e 0 t ne O !< ,ia , - k I av , kn -h nóoto < krt η n ” p ,< p tg ro o >\Bz, heo. fehue lm egv ote.fekks for hooloto kodxto sec'', tied peid ml <-\ö%\okho>< s nxgR-izlo ;,ö\-v _; from ore η '. »1 ) „v. d ' tA ·, í tb\ FU <»''si)λί' \ i\. η ót <T, VJ s, est \d\ot ^! a\ rth the knowledge about the variable domain of immunoglobulins, many attempts have been made to produce alternative spacer molecules. [Sketra, A, (2öőlAníicalins: a new eláss of engineered iígand-bindiog proteins •oiPi notihocty-llke propersies. Rev. Mól. fóotech. M, 157-275., Xm L, at al. (1o02) Oirected evohrtlon of híghaífimry aotilrody mbmes usmg m-RNA display Cbemisíry A. biology V, 975-9-111 These molecules are able to bind the target protein and are surrounded by a carrier, i.e. they can use the signal; smmnnp\y',p.{,k tnods used by the invention in ro-k \. - yen lmu-u eh'-nen- ro keto ntelem.kB rotahö) leiV/te-,.' in the sustainable future, which may offer better alternatives.

However, it is known that antibodies and antibody fragments are modified, for example, by stabilizing them; wrzmjt (which we call ídtskmcron .rntPesroek révcíup révcíup l gy lí* reacts especially in the case of a ChlF reaction, the selection of the appropriate antibody is critical.

YES

It's skiable. The accompanying IP requires an antibody that recognizes the antigen carrier, which preferably binds protein A and/or protein G easily. The use of a specific -m-io\ μ v es is characteristic of Chile. pdoánl n.tnot above tiun means au aeíomaitkusun that the mii-na CníKte ts< applies Few antibodies are sensitive to the inhibitory factors present in ntput kroms nnntábsn, which result in an increase in the binding efficiency by increasing the topat mcnnyfour. In general, polyclonal antigens are characterized by multiple anti-clotting antibodies, in contrast to monoclonal antibodies. which recognize only one eptope; both have &?. advantage in immunoprecipitating tnódssssemk».

MlmöléTükk;

The m<!'''W.< ». »/ , 'í <wx < s ,uoyn >/ ovo «κ 's ·,ο>, elet' dt 'k les ' v ei< t ,1 e tte ed ' · ces presentation method such as phage display { broad presentation) method is used to present a peptide library for an antibody of the investigated protein, Bg in a preferred arrangement we use a random peptide library. The length of the peptides is, for example, at least ?, 8, or h amino acid long, and at most 22, 20, 18, l. yes, 12 or 10 amino acids long. If the consensus sequence is known, a smaller library is sufficient. Similarly, if the segment or part of the peptide is N or ¢2, the variable ^suffix will be

Then, non-particles defined according to the invention, viruses (virions), e.g. bacteriophages ·· a library showing the library of pepltdek - we provide / set up rain or shine), which contains the appropriate coding nucleic acid, taking into account the degeneracies of the genetic code

The library is then added in several selection steps to the tnofectda (for example, aítlliest) that recognizes the elements in advance. The recognition molecule preferably has the specificity of a known gene and/or the target's initials. nvz te „ kó'o te-te a 'e ^! <\n comes to the molecule and its isolation to the bound particles, and if necessary, some form of analysis and monitoring of the enrichment. Enrichment can be observed during the selection steps. , rt,no>es^ev'-n,kn ! o,K sa$,v \t - a»ct tv nsk detection. Alternatively, nucleic acid hybridization to a ChlF containing a large number of suitable sequences can be used.

The mtmotope clustering method can also be used in cases where the protein has a suitable epitope. ϊ

A walks &< >. , we can also use evolutionary methods with tendons to insaturate the relevant mimotic tube. In these methods, viruses can be used and propagated during selection breaks. Mutations could occur during the selection steps and viruses can be extracted from the linker presenting the ephöp sequence. The viruses obtained in this way can then be propagated into a suitable host organism.

Epltopes, as parts of the molecular surface of a given molecule, ^and nshnotopes are also the following:

In the IP method detailed in the invention, a library enriched with the appropriate epitopes can be used, but examples of use include nanoparticles, viruses, or members that contain the appropriate epitopes.

The results of traditional immunoprecipitation experiments are analyzed using immunological methods, such as Western blot or ELISA. These methods enable detection at the protein level.

Λ's invention has been validated by EUSAs, as illustrated in the examples.

In ChlP ghosts, the nucleic acid-binding proteins are immunoprecipitated with the associated Ö'NS-sd or kNS, and its presence at the specific location can be measured at the nnctelic acid level.

Methods can also be used that identify highly modifiable enzymes, provided that restriction endonucleases suitable for this specific modification are available. An example is DNA adenine nusduan.'ímaA ídsn* u\ I z a t'hcu <- f!\s nduroud mchnifa, in this nt'\’ve<hea s dam-t &

the fidváfier fidváfier fiskmahok express at the fusion half, which methylates those with adenine. which are not covered by the target protein and these parts are identified with endonuclease maps.

This stable, robust and fast detection method is qualitative PCR (QftCR or QEC'ft). However, with QPCR, since it requires a known sequence printer, it can only be detected at a specific location.

Another method based on momzh.ueMO; a t'hld on 4 zip, vas-p. í biPeto?·, mord uses nukiemva dynamic design atray technology. The sequences found on Cdlfi depend on the purpose of the experiment. They may contain pre-engineered genes or regulatory regions. While this method is relatively simple and efficient, the resolution capacity is quite low and depends on the hybridization parameters.

Sequencing is also possible after QFGR measurement from the point of view of Chifi detection.

In the case of capillary sequencing, which is a fast method, a sequence mixture is obtained. This can be used either \ tIP ϊ κ ió v ~s vg >tv skuó·- v mo. Oot 1 ove^ é nloí nuó b td no u u>'í '' a Cdlfi-see ktserfetenet.

The Chlfi-sec experiment is the sequencing of nucleic acid fragments that are coprecipitated & specific probes! with the protein of your bones. In this case, any precipitated nucleotide sequence can be confirmed, and the result needs prior knowledge. [Kczarewa I et al. Ampliftcatlon-free lilammá seqoenemg-iihtaty preparation íacilhatos Imponmd mapping sod assembly of (GrCj-bfesed genomes, Nát Methods 2bl)9, 6:291-295., Gorán A et ab Cteuatm profiling hy árectly seqnenemg small qnaritifies of iawtunepteepitated DNA. Nat Melhods 2fiiö, 7:47-49,, Bsrskt A and Zhao K: Genmnie loc&tíon analysis by í.'hlE-Seq. J Dell Bloehem 2009, Iö7; 1 May What method is available for interpreting the results, yet there is a serious potential problem in the interpretation and data analysis of CisiP-seo experiments, which requires the careful use of available software?,! necessary. ILdt ET, et al, feXCA: GldP-sep fechnologtes and the sfydy of gerte regnia-dm, BMCBIOlogy 21)10, §:5ő, Nő, DA, et al, Entpirieai ntethods fúi coaholiing Ibise positives and estimáikig eonfidettce Irt ChifiSeq peaks, BMC Biottformaties 200b, 9:522. Pepke S e: al: Competatioo boy f.?fil fi-.seq ami KNA-seq studio. Nat Melhods 2O0 ⁴ k h.922-652.1 In any case, the quantitative analysis of the selected Cihp-sec peaks is recommended,

The controls of the invention can be used in sequencing reactions and thereby provide an additional opportunity to control the quality of the CldP reaction.

Who:

Based on the invention, a kit contains controls that can be used as controls in Ifi experiments. Even more sophisticated kits contain positive and negative controls for an ifi or Chifi experiment validated with a specific recognition molecule. Preferably, the controls are provided in solution Ibtmáhan. In the preferred arrangement, ¹ idvnui 'mk\Jx v <í ni,'} ^k '> í ?„ g efe m ,hb >. t<. ím„,, xí lttt»>ox ziijlba essay identity Is. or at least those printers that meet the requirements of the epitope region. A preferred é,n.ndexshén Όνό'Ιη kooto'boá, mmi mAlad mm \x-ttt\k»u. .intíte'” knotfd mk, grew' ípt I Bring ee>eb non-negative ncan specific .kootrols ;s ensures the kit.

zen P'kll ip pt.rp-te-, Vt K pnuerek, movs<'i4x0ok, tapukn nbkaok e\ enub tx.taeusx-\ n. The application of the invention is not limited to these examples only, as other methods based on the invention can be implemented by a specialist.

EXAMPLES .4 ÓK pwpíófí aíííítíkó ÖAA s reá vónom Afóaosdsíz A//5 AT vate'óa ésveator sepp<v'Őzfeö

MI3KE Pepiidé Phsge O-spásv Ooning Veetor f bought New Loglaod Bíoíahs lake í'lpswieh, MA, USA (NbB, BSIOlSj). The DNA sequence encoding the P4 peptide is cloned into the M13KK vector, the M13KE ok,! ?' í K x ' íg /op' íwxtl 1. oo b. 1 >''''é Wx'Uite ,xx i tM L 1 k oK> χΗ'ϊΑ oí, t csaUxS based on instrtskoiői (Ph.D. ™ Pb&ge Display Ltbrarios insituoimn Manual (NBB, E81GÖ}) the nxXk described subsequently -easx!kka! V x.LíO^ mAunxóse egyxxdu ozgooukleondot xt *n$m>a kkbnh iootwji xzonetwdm, smmis are coded by P-! nept-dct Fig. 15, and in addition contain overhanging sequences which in a later step e ,os< gmk,i resulting daptasvala'Kígixtuklcx'tíó kfenoxtsat

S^CATÖTTKPGCCaUC0C€A0-nTAACTTCTTCTTrAACACC1TOGCTt'CC0t3TrrrrCrfCzW.A€r fe' VΛ \ V1 λ k-fen -: fe O í li'<i '<Mofe.po }t _xt Pbx _b fx p. _w x a'

The matching oligonucleotide (4.1 µg, SdOpmol) was attached to the "Phaga Pepitek 4 oligonucleotide (Sug< - ISO ptnol) in a 3a molar excess, the sequence of which is identical:

$beATGíXOG<X)TA<XTrFCTATtOT€-3 ^< (STPÖÖ997H?, ,exteosion primer''}. Alignment was performed in TE buffer hsr containing SÓ ni loi)mM NaCb, heating to 95 Al and heating to 25 Xfem for 30 minutes. The linked oligoookieetid pair was extended with IS U KIonosé-fragrnent 2ÖÖ ulNEBuffer 2~ixm, which contained KI mM dK'EP mix. Initiation was at 37°C for 10 minutes followed by a 15-minute incubation. η '7 C*c ' x».n ,tsw,-,),; bp x'\ rx, o·,, ixo gel analysis was used, which was performed in Sbá-os MetaElmm Agarose (Cmnbrex) gel using a Slo-Fisd Mini SsbCell device. We cut out the 71 bp fragments with sticky ends, and then the Dilute Pere Pt R Oeanup sheer Mt tR-vhel bxtaznáhurk. ;t DNA concentration was determined with a Nanoferop spectrophotometer <25 ng ·' uh. 4 µg of double-stranded MIÖKE broad vector was digested with 50 IJ and 50 V of Aoeoli restriction immunonuclease in 50 µl of ^NEBtiler 3 at 37 °C for 5 hours. The linearfeal vector upland Kifeos ”c esot'x'd Mo!e<cfe'Ktx'locskganvu.ζΒίχ>-Κ»χ5ΐ, .xa grabs xem tmgnX'tdx't t50? bpl fe--tgtnk gellx-i and then GerteLlezo Kii-tei íQbiögenef purified, the l<;ncernration of the DNA was determined with a Kao.ol'.kop spectrophotometer (200 na/ ti.ll tneg.

Vf and, xO dU'i \>.t ,1 > P! < CI '}M 'K' MM» í >O , O _} s! ₅ jO , _/v! ,g _iX

1.5 ó é< to I 14 gt íP oix xó telr ss\ 1 s,o, ikd, s I,? I fe>'<\z poí etat 'á C v eg\ ét ét suka·, The heat treatment was carried out for 15 txtrc óóX-oo, In order for the P4 teódedb sequence embossing , χ! '<' }χ \i 'kt if S >s>, . A < \hi v es \ g, <i'>' ο <η a (, „ \1 L na lse s „'m cicgsct ck'kts,>pm hasszl sz strain ER273S is transferred to Etií. ik.1 ul ísgátssnsöi efegyiietíon lOOol eívíktT<^kí>tnp<-i^?k% SR2738 cell! (pre-manufactured at the supplier's suggestion), for electroporation BIX .'is.t-üopm Λ»! e·, «.* "me\ keséi;?,s :w ^! !iSukh (2 ^kk Rí SH1 X\Ct tt-ph X nh?ge 05Y\ -.eusmavc , eharging: 1.4 kV. restslting pulse íength: 5 msee). Λ into cuvette 1 ml of SOC media was added, and then the electroporation Xs were transferred into a 1 ml flip-top falcon tube, so that the phages could be completely extracted 45 minutes after inoculation with the safto S /'X', 250 spso svhe^egd test veNzsporifás). löt) tsl fuís porsiat·: ? Mix with ml Top-agaí (!0g. 8aeu>tryptone, 5g acetic acid, 0.5g NaCl. 7g Bacto-agar/L íSigma, Oennaay}} mix, then a5Ύ.'-οη the mixture 200 ui h,R273k kísdáfai 020 ml) contained, the ash was previously mixed (OD59S: 0.05), in LB medium This 3? It was carried out at a speed of 240 ^rps . The lop-agat mixture was sliced on LB-agar plates containing 1 mt/t of iFl'G/Xgal (1.25 g of iPTG and 1 g of Xgal in 25 ml of ÖMF-bsm IStgma, Germán*>). » kás;s et iskubetó 37 ® Cmo, mixed breasts, Betagalactozldase induced by 1FTG dlt from the functional M13KE vector. due to expression, bacterial colonies containing distinct blue lags appeared. These are done with ^dessl tips. After afsiibíotsknrn-srsersting iB>, then with shaking Q5Ö rpsu was grown at 37%' for 4.5 hours and asstphfikalmk until each Ó. outstanding. Large amounts (1-4,000 g, 1 egg) were removed from the culture medium, thus helping the proliferative strain. Boesse. For O0CR-based dig filtering. The jckmiíes and sneafcielö vesiets of the inzest sequence Q0CR in positive kionos ^„PS x ' X,, SA'bs,'<P< O 5 '', 5 < D\ i,>it ';<·,',·, 5 {'θ' DNS 1 I Sx'SVÍt'lx k'S'lá'k«f'' V. .i'OC Μ13 -Oá sequesUtlő printer A8I 310, Avasst DNA sequesssdo machine t3A and 313 fig.s, The posiitv F4-MI3KB wide kiormkat, which! During the QPCR-based screening, we add 5 aliquots. The "aiigtioCs" were taken from the tssnpbOkated woody trunk (see below) and the one from the early hanging iázisbas (GD5SS: 8,ÖS). For culture ER2230 |2ö tssl L8) ssdstík, majó istkebálitsk 37X'. ^: tsst 4.5 hours of digging, including shaking (250 rptss).

The culture was then centrifuged (000 g> for 20 minutes, and the half was treated with 20% FE<s/2.SM NaCI buffer in 1/6 volume. After incubation overnight at 4°C, the complete fugat, centrifuged, and mosbsk, tsttijd 50 ul ^: smazuszpestdálttsh in T8S, bogy we get the tsszs Itón ampl itskalted tag kidsstsktsts for further experiments.

/•>ísgj7seí3<' e:/ötó.?.háAi· árrssvöft'íí /es/ííúnps'ezdp/fífe/óA.o? í'CO/.Aí

223'1' cells were cultured in DMEM, 10% FBS. Penfeiiltn/Streptornyein, Glatatnm, in a medium composed of 5%o$ CX)> söus'i.tüt> s ’< ^'íxzíííx μ Ο,,ϊ '' X! p u η. ancient S korosé zt.v s i2'> Ό ί,κΑ,ν,ο t^hínsOiO sejs/Oaska),

The culture medium was removed and filtered with 20 srsi FBO-rs. Add the remaining salt (Sígís-ask and let it sit at room temperature for 10 minutes with gentle stirring.

We added a further reduction of 1.7 and 1.07 Mos gesso to Isozza in order to fix the joints. Put the flasks on ice, remove the filter as soon as possible. Wash the flasks twice with 10 ml of ice-cold PBS. Add 5 ml of ice-cold 08S h.tiá.-'ara. Collected cells were transferred to a 50 µm conical bottom flask and mixed with 20 ml of OBS. The cells were centrifuged at 1008 g (4°C) for 10 minutes. The cells were carefully removed and resuspended in 1 ml of PBS:

and like before, 20 ns! We washed with EBS. Et-3S was again removed and resuspended in 0.5 ml of ice-cold EBS. I place this in a 1.6 ml low volume tlo« Pmrö oukíoeeoitdkio.sötek. The cells are centrifuged at 4°C for 2 minutes (6120 g). The fel-'yl-Izot adipates and the <.',ipedeks are left in 1 ml of sonication buffer (IH SDS, 10 mM EDI A, 56 mM mik k pH 8 E with pmtemaz inhibitors (Boche, Switzeristtd, Cnmplese mini 04-693-124 -00 EiA puff 15 ml conical aijti ptsltsphire was transferred to rain (I3Ö Falcon 352.095) and sanitized on Blorepfor sunik.utor (3siö pere, bfigh setting, 30 tnp pulses)

See the sample. _s 6 mi low binding) tubes and cenirifttgulat 4X for $ minutes (Í4000 rpm). The fractions were transferred to a clean microfuge tube and 50 µl were used for fragment analysis. The residue was stored at 0°C.

It is the fragmentary artalizt. performed as follows; the 50 u) semi-floating, klegéssitetut 200 u! with elution buffer t irisseo dissolved 0J M AailCO.3 and 1% SOS). Add 5 M NaCl and 2 µL 63 M BOTA (pH 8.0) and incubate overnight. The following morning, RNAseA (stock solution lOas/hl) was added. u « 'C e vi.o L.U'.'ie» < t unt >ip 1« o Ml 3 \ . tpb 8 <'t 8o I ski '>. V v> e, as t ^s > Iheaen)?.·.? ΚΗί^'κίηί hn rnoms t <A tw^o ni·,; AI >\ r . v .vdehn ibemonisMx·' it koö.ftnk m.,!'.- > kOzhen (1600 fpm), in 2 hours and at dS'C. We completed the -iszthásS Rocht-: High Poré Tempkue Erspönthon i\'K stalls. I cut the columns into k, -szar H) ski parts!?, to reach the 100 ul eiuáturn. Λ mennv oeget Emim High Emotiviíy Double Stranded ION A Kit-tei (inviirogen, DSA _; Q32.85I) was determined The concentration was in the range of 76-löU ugAtl

We examined the size of the hogrneut with the help of Ágikat '2 lOOBioanalyzer and 2500 DNA chips, which are installed in S.áhran.

Cő/E á&ar/ef

All steps were done on ice. We mixed 264 ul Erotem The pearl in IInvltrngen. DSA. Dynabeaás 100.020} and 264 ul Protein G beads (lovm'öpen, USA, üynzheads IÖ6.64D), and 1.336 sd IP reaction was performed, which was prepared from 1 9 ratio of zoocizing putfer and IP high puEr with the addition of proteinase inhibitors. iiP hicifő potfer: u.öEE, 8DS, l,HB-ns Trtton X-100, 1.2. tnM Ebi A, 16.7 o?M fris, ρ|Ι8,\ i67mm NaCD Centrifuge this for 5 minutes (:6I g.s. The íeiilasasoi were removed and added 1336 uf IP reaction poffer u),vp:i maét csmir? your joints. The superlative is the basis of the previous ones! we cut it off. .The pearls are added for 528 ni puffs. v. after & his pearls separated into two equal parts. These were supplemented with 1.4 ml of reaction buffer S'· 24t3 i'.DACi antibody was added. to one part. to the other where IgG (corner). The tubes were incubated on a rotary plate (2d) rpm) 4H'.Eou for two hours

We placed the pipes in a magnetic stand on the edge of I, and you removed the feuila salt. Since then n»8A« to rain. For 528 ni reaction pofi, we ttöturtk and the contents of the tubes were further divided into 8 ui&caony bandages) rain I A\t e.en. i 'S A, MCT150-LC, 3i I-09-051) to HDACI (Diagettöde. Belgium, SNI44960)cs uz igGbez similar to tyboi nevest pearl/reskciö).

The 30nl ultrasonic sonicator is the one in the bottle. Ultrasound-treated krotnattntn of 5öOK cells tenfold htglisdfunk IP grower puffs (and profeinaz. iobibiforokkah slkavt? i mM end-cone entry DTE-vsi) to áOlbd, This was given all- pearl tortaImazö ),6 tni núkrceenirslttgaesohe. In the individual tubes, Iev6 reactions contained their teeth in an appropriate amount, as shown in this example. Signed by Table I.

We incubate the pipes overnight on a platform.

1$ ' a ν'-... a s n ,bz x> \>.\ ·κ fs t> i. I u'«, » c ^Í> λ s.v roots éOOitl tiétéu ChtPA py berrel motos (0.1% SOS, 1% Triton XiöO, 2 mM ΗΤΤΑ, 2.Ö mk! TrísHCi. pH M ι-s'j M \)t ·, U! 't (),''í\»l· 4 .,! ,ΟΙ Mgv p v'Um* v'« tgk with Chlb 8 powder (0.1% SÖS, 1% Triton XlOö, 2 »M BOT A, 2Ö ntM TrisHCI pH S.1, 500 mM NaCl and protease inhibitors), and with ChlP C buffer (0.25 M llrimin chloride, 1% N?M0, 1% nandium deoxycholate. 1 xoM LDT'A, 10 onM TrisHCl, pH k.l, and protease inhibitors) and then twice with TE powder (0.1 xnM Ttis, 5 soM Hiky pb.S> \ Cvvmwkei re-made etklk 4tkd 1E potferpen vprotctnaz inhibitors nelfellT and» put them into a clean tube. ?We placed the tubes again in a magnetic stand, then after an egg & semi-lunch I cltotmm. Hitei a from the point of view, we played on the »z<^aöómm»ckfet.

The pearls are shot by all psttferKm teKcütlk. in a copper machine (HX1ÖRFM), mknbalmk at room temperature for 15 minutes. This step is repeated twice and the results are combined to form a pure _{oxymicrocentrb} . DNA Isolation was carried out, as it was performed during the fragment analysis with the I0Ö killing protein of Dbis.

!MM.RÍ?bOö<iö.íbk<Th^^

HOAk'I ntoövktnüuPs untuest nnmonogen epunp pephd kvdolö sectenc;,ö,r s/nbklomvöik Pepink' Phw ihs-pUv Ck'tug \ ,í/\ az v^ktörR b.'gy >Ψοο reconRnam Mi'ki members.Hlmmok Jo, amei> sotdepa ep.mp pepiül k sermmohs \egerek iuzsojst the wide bo-oktokéiieseseE of Geneva the igniter ηο&Ηζί,ο Japgm e.z 'snreoegöo epuop pepnd ^ek^oncara; a 1 (H; , nmnokiorah', our artikvHtc? szstnaikink, bee,; the middle of the Diagonodo; NHhV 'EEKPEAKGVKEEVkt A-COO- (hereafter P4). The rntkleobd sequence of Λ R pepiié and s/őoo was discovered and optimized for b coli Who's code (with use, which the oviit source code x»e'ei\'sp s,A>. i «μ fichaöP , g;, sm> ion

5 ^, -GAAGAAAr\ACCCATr\AGCGAAAGGTGTTA.AAGAA(3AA<5'rTAAACTCGf.O-3 '(Leg).

.The HNS sequence of the above eotlüet was prepared and subcloned using the Agcőei (5') and bsgl (3*) restriction endonuclease recognition described later, instead of it, the genl?t-bo! with the parent coding sequence, the 3' secretion signal; sequence and the 11 protein-encoding 5' sequences of the mature gene. As a result of these steps, the R-geniH protein of fermentation acid, as an N-tetminoltly feziomtlt peptide t'Pó), The Rgonll protein .)''>' is copied for incorporation into the mature ΡΉΜΙάΚΕ broad, which the E. coli Ki? trat-sybrinal precursors produced in cells and derived from .R-M13KE DNA (Figure 2). After the purified DNA fragment containing the R heptide coding sequence (see above) is ligated into the MÍ3KE vector and transformed into E coli KI2 cells, blue line; Bacterial colonies appeared on the LB-agar-ÍPTG-Xgal slide, which was covered by a bacterial lawn, which showed that the mucous infections of the wounds resulted from the iigulation reaction. The individual MlsKb member clans should be propagated from the fire signal slkiliömilö kök colony, and between them! ó;öi (plaque No. R 2, 6., 1 L, 12., and 13.) we examined the presence of the R peptide coding insert signal. QPCk-ríd, Ml.) specific "ibnvard primary tCencrai E or S). liléevé R,.insert ' specific revert: primer (Pepikled R or PoptnleA $) and using a general M13 probe (General TM) During the screening of the insert encoding R pepiide Msed in clone 5 (No. R ií, ö., I L, 12 ., and 13.) turned out to be positive. The DNA sequence of three M31KE broad clones (1M 2, R o, g$ R,IM was analyzed by capillary sequencing, with the help of which you can determine the correct origin of the R pepnd coding gene; and its reading frames. All three come out; (?4_2,1M 6. and H 13 - its sequence is shown in Fig. 1), the Hnzitiv R-M1 5KH phage clones IR. 2, R 6, and P4 ki) - as the entire length was 20 atnpiifkaliiik and itizttioft, EEG ; Precipitation of NaO.

n/tf/71)4(2( mormk/OTífe öííP'/«???/ á/mJíféyt?.

For the use of phages as a spike control trend, we have tested that the specific HDAei tnonokos it a ,t <\t, jo , \e \ n t >o\x,

IbÖ.ÖÖOO copies of the cloned phage liasstälmünk 1 ÖÖmicrofitter ferfangstö ChIPpyfiet and 3 ni antibodies. Chromatin u · >, ⁽ prtu !..! 'k< bn · '>\ srínvk ί íK n< t\ m rt , vCá2>,·4> t can be found in the detailed protocol sz materials year methods section0. In short, the antibody Protein A's and protein B's in the ratio I: I were bound to a medium containing coated parasuspended beads. The excess antibodies were washed off and the beads covered with the antibodies were removed from the fmgnteotáh derived from the fíragmenía lÖO.ÖOÖ HEK293T cell. Vt , \C íi, rt k 5 4 < - Oíc nk tt's'lkx. s<5' te _s s -,U 5 | dt 5!

and paííerhen containing LíGl, nttptl twice in ΊΈ powder. After the washing steps, the knitting! complexes entkcios ptP\ bet e u.'tck , i vutz \kí\,.'\'->t<·, <ten. > p, p non \o k vmiseo. < tte » V\$ ο-, lo \>s ö-.rt íöxí G;,. we cleaned it with rasrtt. Λ recovers DNA-; Further details of the PCR procedure can be found in the materials and methods chapter.

Determining the fraction recovered with antibodies from the guild .^zehasontttotnik the enrichment is. iop-justl, and we found that the 3ogltdac 1 antibody is able to bind almost the entire input cell í,\( t.i, (k ( A '?>, 'W i,

After that, check whether the virus is able to bind cloned or empty phages. I assume that the system is working well because IgG does not bind the cloned or empty vector, which is not a tax-relevant application in the QRCR photocell.

Kitmpzptuágpk

The QPCR assay was designed to detect deionized phages. One of the primers and the probe is specific for Í5Sg, while the other primer is specific for the ionozvit fiagmesrte. Importantly, the QRCR essay was designed so that it can also identify iifcs members, such as: negative coaholics. See Figure 4 for more details.

We have waterproofed the following copies of our facts: l.öíXl.bOö, I0Ö.ÖÖÖ and 1Ö.ÖÖÖ. Now the 10,000 copies were also detected by the QS'CR essay, s.z msed V'M'xo ο-, the wire principle M!3 lagan; also a recognition essay. During the apparition, we believe it too; that the antibody, oőugp, mnaovav s.tu, or any cotnpo·· nense of the system would not bind to a determining amount of negatv hone! shirt

QbCik/Aetekíóias:

> out! oȟ\ . 0 01*' 'R essu- t .ha&znasnmk:

1. The QRCR assay of the cloned log: used a single probe and a general tbmard oligonucleotide that binds to the backbone of the iüg, while the reverse primer is cloned •teertte-specific (Fig. 4j,

Peptide R gCCgACgC(2Aj?'n'íAACn'

General S íFWt ATTCAk'Ci'CgAAAgCAAgCTgA

General TM BAM-AgAAAggl’ACCACI'AAAggAAl'TgCgAAT-BBQ

2, Mt'ke laz _; \!t \t; xko-u cs hit t '<o 4 λά o p'oba edt^n cz case I n \C!-4, tTG'v * b,ts (Roché) trial number UEEAk

MHKF kl'Pl tg f A < ΛΠί,ΛαΌίόΤϋΗίΙΠΠΛ

ΜΙ3ΚΕ A UFL40 REV; 5' - GGCGATI ÁAGTTGGGlÁACG

3. All cloned Thrush-recognizing QPCR assays: use probe 1.1RU21

Rep. í·'' A t }'i ΓΛ '' riuCl Ali. C( RGAAÁA í'G.MAí Pepiidet-5 A URL?! REV: >’ GTACCGCt'ACCCTCAGAAC

2 µl/ml DNA of the cleaners was used in the QPCR reaction. All QR'R. measurement was performed in triplicate eh Ez AR n sktn λ ke., \v>xet r 1 rum H oh ! t ski-u'Cs u? ¹ 1>\ eCO p 0 e Mgf 2 ripen! : 2 η V \ í ·,»..;· ti/h h>ss<. ? e et ι ^! 'Mu r <. ?<'}> Jt.tu «(\ _v o'bi's tt <t όολ (2ötj«s), 0.13 ni Táp Polimeme (óti/nl Fernsentas .KR 0402). The measurement was carried out on the Rtx:he Ltghteyder 400 device A second derivative Max method was used to calculate Cp values when analyzing the results of the QRCR reaction.

Panels A and B of the figure show two biological replicas (replicas), according to which we can see how much alkali is extracted from the C'hiR reaction salt at three different concentrations of HÖACI antibody, namely I mtibo, I9ó enr s v tó e.e- 4 C and 1 > panel shows the amount of members that we add to the system, i.e. the mput tóM and the te-tes ! Kamts-ég } Λ <,h\ R into rigids? In the case of an IgG Lvtroi urn, it is negative - On panel 13, we can see that the empty MI3KE members are not bound by the HBM antibody.

{' '.-for k, t in Table 1 and insert the diagrams of OF.

I. To tabulate; The api ke QRCR reactions evaluate Cp

r. βΐ!!1!Ι wrote Béu liiiiiiil iieisiiiiir Bárt M a Ai 29Jí e §0*6 P4 ~ HÖAC1 21.30 Fake 293t ·:· .10% R4 * phytJACi 21,32 21.31 0.01 AJ 205í r Ι0 ^Λ 5 R4 ;· KBAGt 21.30 A4 293f K1B R4 e KBACI 21.72 A5 293te 10% P4 4 BiMCl 24.72 21.03 0.12 A5 20Jt i 10% Rá : HBACi 21.52 Price 2931 ·:· HR'5 P4 s- INPUT 40% 23.41 A8 293t e 10% P4 4- INPUT 10% 23.32 25.44 0.13 | oh 29.1t s- §θ ^Λ & Pó * INPUT ARC% 23.35 Bl. 203tr W-\5 Rá s-HöÁüt 25.00 m 2931 e Ki ^A 5R4 F .H.IIáCI 24.95 24.95 ο,ιι 1 m 293t ?0%R4í· BBAC1 24.50 Boo 293t; ΚΓ5 RÓ * ttióACl 23.74 B$ 203t r 10% Rá ·;· BI3AC1 25.75 25.70 0.00 Be 2931 4 §0-5 P4 r KBAC1 25.50 1 B? 2931 4- Kt ^A S R4 i INPUT 10% lllill!''· I BB 293t r WM P4 4· INPUT W% 27.55 27.5? 0.03 j B9 293t i hms w ; wur 10% 27.52 Cl 2931 :· Kt 'ó Ró ttOACi 28^22 1

€2 293í -i- iÐM F4 ·;· HOAC? 28,33 .28,25 0.07 C3 2931 ·}· HU 4 E4 o lt-í>AC! 28,21 <24 293t t 19 ^Λ 4 Ed -i- HÖACÍ 28.3 d 2931 ·:· Ki '·4 E4 r- ÍÍÖAC5 28.89 llisllllllllll: 0.24 She 203t -r 1ό ^Λ 4 Ed 4- HDAC2Í 28.03 <27 293t 4- fö'4 F4 r TENDON HEAT % 30.72 €8 2931 r ίδ ^Λ 4 Ed 4- TENDON HEAT 19% 30.07 38.91 0.10 €9 293i -t- ltE'4 E4 ·;· TENDON HEATER 19% 30,43 Eh? 2931 -t- 1<P ^V 7 Ed b TENDON HEAT 18% 21.70 8.8 293t < |9 ^Λ 7 Ed 4- INPUT 19% 21.71 21.78 0.08 E9 293t -a ÍŐ '7 F4 r TENDON HEATER 18% 21.88 F7 10 % Ed 22.73 F8 iir <i Ed 22.00 22.99 0.00 E9 10 % Ed 22,33

We also observed the ratio of the input fSg connected to the 3 ng ssstltesf. 2. In the case of 1,000 added phages, this ratio increased to 90% <222,780 recovered phage vs. 22,731 non-recovered. ,

Finally, we used mouse monoclonal antibodies against MhC12 and E300 protein as a target in a heptapeptid phage display library selection protocol to enrich the broad range of genes whose specific bindings are able to target the antibodies. in order to produce a broad spectrum of chloite pcsni that can be used as controls for the MB3E2 or E?00 ChlO protocols. Why is the random seed library Pb,ló,-? Ehagc Display Pepiidé l.ibrary (MED) Itaszftálhmk, atnely such recombinant MIIRE phages that contain s hanging ghetto hutovte ^l cu \ s _s ' mk, v, suk <>/t< \ xn, c -v -,/smAI χ * , *\|i όζ,,Ι v, d ko ' tat are displayed, and. Library branch concentration lö ^: 'pltt-'ml, in which all possible sequences (which "9* o,„>'us ίϊ piTOlíxa-'·» d'> tsprs»vnt.\ o abo' e aha o J , f bso Af>te~\'>'k's goal this was done on antibodies in solution as a first step in a broad blocking step, which prevents non-specific interactions with the antibody, and in which it shoots ni aliikvot as input (in which the results of the logs: number 10), pinj 1 ml of saline was added to TR1S buffer with 0% Iween-29 containing l%RSA, and this was incubated in O C'Oit. vr te -sp Ml CE2- t.ig>p^'ó-spvCthkuscsttsk-ste', ad-uuls to the mixtures and they are incubated with oö perseg shaking. The capture of imrotmkoinpie;o?s ta precipitation) Protein ü was done with magnetic beads el fDynabeada), with the addition of 5 seeds (1.5 mg) of protein and magnetic beads, produced according to the manufacturer's instructions (ctye&headst), and this can be struck for 10 lg at room temperature. The beads are packed together using a magnet and washes in 1 ml of IBS containing 1% BbA. Shake for 10 minutes at room temperature; This is then reduced to 150 ul 1 ntl sM rnsók'kd.d iplluJkAz élénk klgok.n,i cotuketo equation aikaíma/cMvah W-merested with W-meres:

enjoy· ·«· os.isnso!·! x. t ;< z..?.l4 x even ahö) by titrating the dilution 50-fold, or the «totum small tneaovies (- i pl), as we mentioned in the General Ml 3 methods (NEB) chapter. The phagekaíutnoí, with the ends of the samples used for the quantification of the member & .the following handbook istoerteíils arttpiíííháifiik; Pó.Z>. These steps corresponded to the first round of the biodashás, and two more; (the second and third stones of the ^{bioenrichment} ?) and which were carried out using the tdikvot obtained from the results of the previous round. The next-generation sequencing is carried out by the 454 Junior system, which: - along with the QPCR-quantification of the ChIP procedures, we adapted to improve the quality of the separation steps and the initialization,

Cblp-altered phage clones: which display a mdkekt mode of mimotope peptide sequences; DNA sequencing has been confirmed and will include ts monoc tonal mimotope comrolls for Chlft protocols.

Then, with the chromatin sample from IO.óoo phage/iöó.óöó cell and Ing examined anraext'ei haxznJiük b'/otat of the previously lein protocol; the member libraries were sequenced using 454 Junior sequencing at the bottom of the standard p:otoko?,; of the 454 .hnsior system http 454 < otr Ámr tömre ny454 kfeenmepüpkmfes-jímlprmjethed-íngnnais/GSJmm;r

Ampkeöfth^ h4tpA454,>

SeyJnoeáö.lö.pdf

Briefly, members were isolated and DNA purified by standard column DNA purification using the Roehe RCR purification kit based on the manufacturer's recommendations. Draft POR arnp isii k»p'd) tbt k ;» for the region of interest of the rodent genome V). k'l' \ <\ Ά >hk ts Oxka! lattana and The ί\..·Κ. In the case of others, you can get more quality indexes (MID adapters) and 454 sequencers for a premium price. The: We mixed the arnpbkons created in this way and mixed them according to the manufacturer's recommendations.

We translated the results into sequences and searched for the best sequence. In short, we analyzed the best sequences of the 454 Junior chair blood donation results (table 2; ads, deüctoks, S'oo ketones > otsu,! reeku-oe takar, .sOiObet, . o-ooefáhutaiSan nuklöotítkska; jarta image uk. e'iavohioít ;k The 5th translation was made with the üsosech software of the EMBOSS package. The results are mimox (.RÓA .öfoógbtvnoAé.v2Ö0Ó, 7:451 dokit),! 186/1471-2105- 7-451) was analyzed with software and the consensus sequence was obtained through a quasi-credible JalVtew representation. The length of the amino acid sequence used by the ohmos: software differs from 7, which is due to the fact that the amino acid sequence alignment generated by the program is not -ed ,r telteim t,.: es 5 ,mm)

2, 1 áhkVas: § egjohh mtnodöp sequences MFCE2 ex páÖÖ anti-protein antibody similarity on aiapjan

ARCH MECB2 mitnmop sequence arch sequence CON...1145 1 1-íis Asn Árg Glu Met Pro He CON BOö 1 Ást- A;a Ebe Le a Asn Asn Árg CON! 1 His Aso Arg Gin Met Pro He GON 1282 I Ásn Alá Tyr Len Ast; Set Arg

CON. in i ι Ilii Ace Arg 01» Val Pro He CON. .140.1 .Dig Under Tyr l.»n Asn Se? Ars. CON .1138..? Phe Dig ;Asrg 01« Tyr Pro He CON. 1488 Asn Gin Ah? Down the As? Ser Mef CON. 55 .1 Hls Ásn Árg GIo Val Pro He CON. . 1730,..1 Asn Ser A tendon Le?? Áss Ser A tendon CON .1121.J Phe His Tyr Pro i'roSer l'hr CON 1555 1 Ser Áss Ser Áss Leo Áss Ser CON. .1327,..1 Asn Aso Arg.01« Under Pro Val CON. J300..Í Dig Row T hr Off Dig Row Off CON. .1128.5 Ser Pro Asn Ars Glu Thr Pas CON 22 ...1 Aso Ser Alá tea Asn Ser Als CON 4225..1 Aso Áss Arg Óla Pro Pro te» CON. 48 1 A,ss Under Phe 1 eu Dig Asm Ara CON. .1149.1 Tyr Áss Atg 01« Pro Pro Val CON. 5545 1 Asn Thr Len Len Asn Ser Lys CON ,1254.1 Vni Pro As« Arg Cin Thr Pro CON 1777 1 Asn Ser He Leu Asn Dig Under CON .24J Phe Ast) Ara 01« Tyr Pro He CON. 149? 1 Tyr Asn Thr Asp Le» Dig Dig CON. .163 .1 Phe Hb Tyr Pro Trp Ser Lyr CON. i 525..1 Calling AsO Ali? Asp Linen Asn Ser CON . 1 No,4 His Ásn Arg 05» Sor Pro Les CON. .39...1 THE:." Ser Alá 1 ,eu Aro Ser Alá CON .2 16..1 Or Pro Ars? Arg Gin Thr Pro CON. 1664 Ast? Sir Ah? Len Asn Ser Aía CON 24g : Asn Áss Atg 01« Pro Pro Le« CON. 5398.4 Asn Thr Len you?; Ass Ser Asn CON 68 1 His Astt .Arg GB Sor Pro to» CON 1359 1 Aro Gin Ser Lee Asn Ser Gin CON 22'1 Dig Under .Arg Cin Len Pro Val CON. .1 S57...1 Ast· His Under you« Asn Ser 11 also CON. .170. 1 Ser Pro Aso Arg Oki Thr Pro CON. 390 1 Ásn Óin Alá Leu Ás» Ser Mer con. .025.1 Ser Áss Arg Gin Pro Pro Le« CON 9 1 l'rp Gly Dig; Gh; Asp Let; Assn CON .H78.J Phe Aso Ara Ol« Pro Pro Let; CON. .1567 1 Ast; Thr .Aia Let; Asn Ser Thr CON 150 1 Aiu As» Arg 01« Who Pro Val CON. .1734.1 Asn Thr Phe Len Asn Atm Leu CON 66J \h: A;.» Sty, Pdu Ser IA<O6 CON .7...1 Asa Thr Oln Len Asn Ser His CON 136 1 Under Pro Asn Arg Cin Pro Pro CON 1648...I Asn l'hr Asp Len Asn Thr Asp CON 1 05 : Huh? Pr« Asn Arg 01« Pro Pro CON. .1 S6S...1 dig Len Asp Len dig ThrLys CON O5í..;i Neither? Pro Aso Arg Gin Fhr Pro CON 1348 5 Asn Ars Asp Len Asr? Thr Thr CON. .1242 .1 Phe Tyr H« Pro Pro lils Met CON. .32...1 Asn Thr Asp Flax Asn T hr Asp CON .1288.,? Dig; Arg: Cl» Pro Pro Let; Tyr CON 95. ...1 Ast- His Asp Len Asn Aro Arg CON .:121..,1 Ser Ass Arg Glu Pro Pro Leu CON. 1442 1 Asn Píis Asp Len Áss Ás» Arg CON 102 1 Ser Áss Arg Gin Pro Pro He CON. .362.1 Asn Val Ser 1 .eu Asn Ser Ser WON. 1427 J Aon Arg's lap Tyr Pro lío Tyr CON. .1385J Ast; Val Aia Leu Asn Ser Í41s on .057.4 Aló Asn Arg Gin Ser Pro Val CON. 254.1 Ast-Ser V»; Len Asn Set Ah? ILON 1420 1 Aló Áss Árg Gin .Leu Pro Val CON. 31 ..1 Asn Ser He Len Asn Asn Ah? CON .5311...1 Act Dig Arg Gin Les Pro Vai CON 199..1 Asn ser Gin Let; Dig Thr Arg CON 1345.1 Under Asn Arg 01» Vd Pro Lón CON. .320..1 Asn Thr Pie. Leu Asn Asr: Linen CON. BSIJ Tyr Asn Arg Glu Pro Pro He CON. 1.371...1 Asn Thr Set i en Aro Set Mei GON. Í29 1 Ser Áss Arg G.lu Pro Pro He CON. 150.1 As T h?' Leu Len Ásn Ser Lys gon 503 i Phe Tyr He Pro Pro Small Meg CON 192 BOW Ast; Arg A»p Le« A»n Thr Ass CON 453.4 Under As« Arg Cin Vas Pro Len CON. 14, 5 Áss Ölti Ser Leu Áss Ser Óin CON .1517.1 Val Pro Asn Arg Cin Thr Pro CON. .1455...1 Aí-n líf;> Oh? Being; Ast; Ser Arg con .05.1 Tyr Asn Arg. Gls Pro Pro Val CON. .374.4 Ast? Set Thr í.e?} Asn Ser Lt?·;

GON 272. i Asi.'Vgbb Is' Pro He fu GON. .70.1 Tyr Asn Thr .Asp Le?; THE";? Dig" GON. 219 J Ali? Ast? .Price Gh? With Less Pro GON. 77J Ser .As?? Sir As;? Juice;? Dig?; Ser GON I306J Huh? Am Arg Gh? Pro Pro He GON 49 1 Asri Thr Under Len Ast? Ser Thr GON ,1400...1 'Tyr Ace?? Angry Olt? Pro Pro tea GON 1416 1 Dig;? Sir Ah? Linen Asn Ser Arg GON 1213. 1 Tyr Pro Pro Arg tea Val Tyr GON .1336.1 Spade Gh? Gh?. Leit As? Thr Val GON PO J Neither? A-e AígViO Pro P;t! Hey GON. .1332. J Dig?; Juice;? Asp tea Asa Thr Arg GON .104 .1 Neither?' Pro Ski? Arg Gh? Thr Pro GON 1436 1 Dig?? Val Ser Len As?? Ser Se?' GON. sit down Phe A:??? Argh Gh? Pro Pro Down?? GON. .1651 Asa Val Alá Len As?? Ser.Mis CON J146J A se Asrs Árg Gh? Leu Pro Les? GON. 1559 i Asn Ser Len Len dig? Sir Val CON Ü.2...1 As« Arg kills;? Pro Pro Val Alt? GON .253. 1 Dig!? Ser Ser Le?? Ast? Ser Les? GON §16?J Neither? Spade Arg Gh; Pro Pro lse GON .131! J He As;?'Thr Asp Len As? Ser CON. 1 169.1 No; Len Pro Se?' Val Len Asp GON. 1494 J As· As?? Ser He Len A;??? Ser CON. .1300 i H ls As?? Arg Gin Pro Pro Val GON. .1925...! .Dig? Thr Gh? Being; .Dig?? Tl?r Les? GON. .76.. 1 Tyr Pro Pro Arg Down?? Vei guy GON. .I76H..1 THE";? Thr Phe L esi As;; Ser Thr CON 1319 1 Trp Ace? Arg Oh? Pro Pro Val GON 1450 I THE:,;; A!a Ti;r Len As;? Ser Lye GON .1615...1 Asn Áss Arg G h? Hey Pro Hey GON. .1,547 J .Dig;? Tyr Ser Len .As?? Ser Arg GON 14711 H?s As?? Ars Gi?? Len Pro He GON 2206.1 .Aan Ser Alá Len As;? Dig?; Ser GON. 99? What Ace?? Arg Gin Ser Pro Val GON. .1594 J Ast? Ser Oly Len Asm Sít?· Val CON 1S9I Under Pro Asn Arg Gin Se? Pro GON 1 502! Asn Ser Gin Les? Dig? T'hr Arg GON .1495.1 Dig? Arg Gin Pro Pro Val Ah? GON. 1.431.1 .Dig? Trp Gh? Being? Dig?. Thr i',,es? GON. 1173 1 What Ace?? Ai'g Gh? Gin Pro He GON .299 1 '1 hr Thr Asn Ser Asp Len As?? GON. .MG,,.! Dig Under? Árgölá He Pro Linen GON. 1650 .1 Ser Gh? Ser T hr He Mis Val GON .1499...1 Dig Under? Arg Gh? Thr P?s? With GON,. .1603! Ttp Oly Ás;? Huh? Asp Lea Ast? CON. 1568 J Under Pro As?? Arg Gin 1 en Pro GON 354 1 Asn Asn Ásp Len As;? Thr l'hr GON. J6S2J Tyr Ast; L.ys Gh? MM Pro He GON. 219 J Dig?; Thr Down Down?? Dig?? Sir Asr; GON .1351...1 Dig?? Dig?? AvgGhs Gh? Pro He GON. ,27,1 .Dig;? Thr Ser Len Asj? Sir Mel GON 1414 i Dig?? Arg Gh? Pro Pro Val Ála GON .3401 Dig? Ser Alá Len Ast? Ser Arg. GON I307J 'l'hr Ast? Argh Gh? Pro Pro He GON. 1718 1 Ast? He Val Len Áss? Neither?' Arg GON. ,1142 1 Me; Tyr Le?? Pro Ace? l'hr Ser GON. .1542...! A se Ghi A la Let? THE·??; Ser Ser GON 409 .1 MM Spade Agg Gin Le?? Pro Val GON. 376 1 .A sp ΑΝ Thr Len Price;;? Sir Lys GON. 4§J IPA As;? Arg Gin Let? Pro He GON. 1311 Thr Ace;?. Hey Asp Len. Asa Ser GON. 1. 1362 Little Ace?? Argh Gh? Ser Pro Val GON 5 g i .Asp Asn Phe Asp Flax Asp Ser GON 1662..1 AT? Ast; Arg. Huh? He Pro Down;? GON. 152SJ Gly Gh? Ser Thr Len Ttp Val GON .1917? Huh? Dig?; .ArgGh; Mis Pro Val GON. 1539.1 Val As?? l'hr Asp Les? Dig.;? Ser GON .1952...1 Gee Gee? YOU?? Arg Under Pro Down;? GON..474...! Dig?? Little Les? ambush; .and s? Ser Thr CON IM15 1 l..y;< Pro Asr; Arg Gh? Pro Pro CON .1530...! Dig?? Other Len Les? Dig?; Ser Thr GON 3S3.J Dig?? Dig?? Arg Gin He Pro He GON. 36? 1 Dig?? Len .Ásp Lea Ás?? Thr Lys GON 374 J THE;??? Arg Gin He Prs? Under Leo GON 21Ü6J Dig;? Val Les? Len Ast? Ser Arg GON .1416.1 Dig?? Arg Gh? Pro Pro Flax tea GON 1324 1 Asr Val Lee Len Dig? Ser Ser

GON 21121.. 1 Thr Asn Arg Gin. Pio Pro On CGN horse?. .1 Asn Val Under Len Asn Ser Thr GON I i 67 1 Thr Pro Asn Arg. Gin T'nr Pro GON 1451 I Asn Ser Ser Leo Asn Ser Gin f ΟΝ I25 j ; Ser Asn Arg Gin Pm Pro i .en GON ~ 1417 I Asn Ser Mel Le» Asn Ser Met GON 2 111 llis Asn Arg Gin Pro Pro Vei GON I Eg? 1 Ser Thr Len Asp He Thr Store GON 3í 3 1 His Assn. Atg Gin Trp Pro Vei CGN. 400 1 Asn Asn Asp Linen Asn Thr f.yr; CGN 209 I Len Pro Asn Arg Gin Thr Pro GON ok?, 1 Asn Asn Ser He tea Asn Ser GON.. 1 >3.3.. 1 Under Asn Arg Gin Vas Pfo tea GONJ.593.51 Asn thr Mef Linen Asn Row Gin GON 1323~ i Akt Asn Arg Kill Thr Pro Le« CÜN„ 1 Asn Thr Val lau Asn Ser Lys GON 11 éö 1 Aln Ásn Arg lap Ser .Pro Val GON 1kö 1 Asn Tin Pie Len .Asn Ser Tb.r GON 402 I Under Asn Arg Gin Pro pro He GON 1377 1 Asn Thr W Len Ass Ser Mer GON 525 A,in Yawn Arg Gin HA Pro Val CGN 269 1 Asn Ser Le» teu Asn Ser Phe CÖ.N 1.491 f Álg Pro Asn Arg Gitt A.la Pro GON 1738 I Asn Ser Leo Leo Asn Ser Pisa GON 19Ö9 I Tyt Ásn Arg Gin Pro Pro He C. GN i 522 I Asn Ser Phe 1 .eu Asn Ser Tin GÖN 53 l Ty? Asn Arg Gin Asp Pro Flax GON _ 342 ; Asts Gin Gin Lett Asn Thr Val GON 1 Pöfe I Asn Mis Asp Len Asn Ser Pro GON 596 i Asn .Kis Giy Len Asn Ser Arg Ot.lN 71 1 Vnlca \ypteu Go Na .Arg Consensus sequences MI.LCP2 Honná 3 consensus <100) p3öb Round 3 consensus GOöi Xaa Asn Arg Gin Pro Pro Ilin Len Volj Asn Ser |Asp Gin) Flax Asn. Ser p..ys Argj

Mira is also iGhafO in figure 9. the nuclear quality indicators reflect the fractions of the branches recovered during the CblP protocol and detected by QPCR. in.

Table 5

Mimotope quality tests show that the CMP protocol sequence recovers phages

Altogether

The soft-touch approach needs to be further refined! We carried out a series of quantitative experiments to increase the relative average speeifte-iias of the mimotope ti the anmestm. The f&g mtmotop communication is one more; as an example for the development of ChIP antibodies, additional rounds (fourth, fifth or sixth rounds) of selective mtmotope selection (fourth, fifth or sixth rounds) were performed to increase the average antibody specificity of the mimote mixing. can be isolated, we can use it to test the monoclonal mtmotope controls of the CltfP protocols.

A < kOóvl ampisliUU tag etnat.'m mkroM felüss/rtitok Nun'.'D Ctob totzs n-eggeseteste o;e\ ko.rpotets V Ά- dew oae?ilr k 5,·« 'Itt Jitek. ι,ν, nk ,t lop v< vnn^ii using íFh.D, ^ΪΜ Phage Display Libmries instruetion Manna! ,N£8). The blue plaques (1Ő-!ÖÖ) are isolated and amplified to obtain unique colonies and the ytnpiiitkal; we test for phage binding and recovery in the CbiP protocol. We purify single-stranded DNA from ChIP-valid phage clones (using any DNA purification kit capable of purifying single-stranded DNA with a size of 7222 bases).

.CMlszekygjgíÚ branch:

Our goal was to provide the appropriate controls for the CbiP-sec tea biology field. In this experimental arrangement, two approaches were used» Mapping of organ modifications and mapping of transcription factor binding sitesV.WV ti'1 \ ,α''-'u< ,0 ogöl yldv A \ ; l\b 'V' oodoó fee, · A no- <. m <« .'öt!,· ,s\ \ No No pia \ 'pin,. ','ΊΛ ¹ K svt Ut t'lb.n >c t't't wua htt ós,\ FiUil'Κ'Νί<'/> with ram antibodies iecbrteS; iOpbkatn!no\ ItasoN 1 n'mtáoáot e'ute-'íek a, í AtlíN geo ptomotttén es N Ιιλ^λ < «miseiéi macrophages (Figure 12),

Chli'-see ktsulelhet Wftx.o,- tv,to,\ fáé, to-m;' Aktot, ko''íreH, ame ,j >;p<edilu? CtdP-ser ktsetWen binds to a used antibody, useful for /something! as a control. k'bir.oiNkyetial shirt-pep; nervous; .ppt.iíng.ization;

The most frequently used ghetto sequence method is Iliamins' "Degveneing by syníhesis" method, in which clonally amplified DNA is sequenced on a segmental phase, which is called with a bridge amplifier. [Chee-Scng, Ka el sl Nexf Genetone on Seeoesetng Teelmologies aad The» Applicatioas. bt: Enology of Life Sciences (ELS). fnhn b· des The Sony ttd f 'Neueste; k;ne 201 «η the ted-PDE, optimal DNA fragment length 3Ö0 bp. This steep tregfeletu is the essence of the í'hiP-tce, ntte: two noklvosroun tmtetesd cuts into one. They found that 3ÖÖ bp-nei longer Oacmcnx n»pn'f e». can·tttmvkt«pctós 'Altmoltt mh, .el»· bet; leim· to detect.

In the evening; aetíOís, et al used two approaches to overcome it:

\<m taltos _; mfication results in adequate size and larger sizes, so it was necessary to improve the efficiency of the printing. On file 12, you can see the performance profile of a partially tragmenized sample, in which high-speed data is used with half the efficiency.

2, The second approach is to use a different fragmentation method. A suitable tool is the micrococcal nuclease method. The advantage of the method is that lmom, i.e. it probably left the large protein complexes behind and replaces the expensive sonskator devices.

During detection of deletion transcription factors using the ChíF-see method, we encountered the following problem. A part of the transcription factor does not bind to f/NS and requires traditional formaldehyde-based fixation. The probable reason for this is that the distances in the complex are probably larger than the hydrogen bonds of this small molecule and Ind. That is why we switched to dsxukáminidií gimarat (l.)SG), which is also suitable for the formation of sizeable molecular bridges. With this method, we were able to provide high-quality Chlfi-see data in the case of transcription lactors. As a positive volume, we used the phage library presented in the invention. The kit used for this purpose is preferred. Designing DSG as a cross-linking agent;

An alternative method of detection is the sandwich Ef.lSSA method, which we also tested in accordance with the manufacturer's instructions. . Briefly, serial dilutions of Μ13 phages were prepared at 0ö, 2.5* 10', 5* KE, Ré phage particles/ml final salt concentration IÖ0 td in the final tooth and added lö\ Kft. , BUSA, EEISA-stripekhex (of 8 holes.) which was previously Ml? covered with phage capmre antibody and blocked with BSA for 1 hour of incubation at room temperature; the wells were washed and treated with the detection antibody. After several washing steps, the holes were developed and their contents were é\l -mt-eu meuhutatoztck 5, oiedntoinen ti' ,mra) art shown hóm a' bt M k soemsck Oi,'<kemwgx. szsgmfikam-an the lake' far particle ad mard Eeuh Et ΗΛ mc-Nctck use.dtea, clap the 'cnveikezcoe stlop'oto· ko - k b'd's- -ra - ' '> eater o e autumn omt , , ό , ne - ki P ml t> is < u «. , ?ra ntáuyaimn, which is already a relevant concentration for the í'/biP output sample, in which MI3 is a broad specific Qpt.’K ? six-.'we knew.

kás.ktayÍtteíipkjmsguáiatajtagyiUUáayos.intetenprsciptlácmbim:

As a precise control of antibody specificity and complex formation, we used a preparation of mature Ml3 KE phage, which either displays a specific epitope peptide, or is similar to the pcc'áhus .nmestre used in the immunoprecipitation experiment, the p'.xed. of the seiekcx- Ά'- án s'Oscktib '&g Jön olögs fi, ηοηο,ηρηχ on the analogy of gteamös makem, the specific ststitest binds to a ISgobox that either contains the specific epitope peptide, or a mixture of peptide sequences to which the antibody can bind (mltnotopes selected from the tandem pepiid file display library ). Á complexes are then incubated with protein A or B beads and finally the beads remain bound even during washing and can be detected and/or quantitated Ml3KB broad: with specific QfiCR or phage loop protein with specific fblSA, or tag bnrok protein specific western blot bowl.

In aliquots of the purified supernatant of a cell count or tissue biogen (cg. Ifi million cells are collected hy eentrifngation ami rassuspended Ιο I mi lysis huffer eomaining 1% Triton X-5ÖI), S mM EDTA, - o m z\„ te; t to , 0 I mái T Y\ n >' s M ) R59 ) ll I < E ~ ö) nhac r n c p , \; < a u' I > 1 ten ml rogy to4cfiimt,\Agy !o Scfiami cm Süoteund, Rt u'umlsagy tó.lt ctű ml, at a concentration of 109ten mi (10.13 pf«/mi by diluting the phage strain solution). The aliquot of the phage-treated solution is rotated at d''C for 30 minutes, then centrifuged at Mofiö xg for 30 minutes at 4X' in a table microcentrifuge. An aliquot is set aside as an Input sample (3-5%) and treated according to the further protocol, which is phage qualiification, fe.g. fór SDSEEAGE and Western hloöing, add egaal vohsme of 2s Iméntit huffer, boii fór 5 min; store over 4 *C for BUSA or QfiCR measnrements). Add 1-2 ug of specific antibody to the remaining broad, hand-held aukots (95-9753) and rotate for 2-5 hours at 9 -'fi - to end the formation of the broad antibody complex Protein A-6 Sepharose (Hanta Cruz Biofech) or protein Λ/Ο magnetic beads (we prepare Dyrta Beadj, for example, wash them in lysis buffer according to the manufacturer's instructions!) for antibody binding (EC specific method); and 3Ö-5I) ul 39-SÜ%?<ί ον x/rcnzsn \i> í\ ni 'fo, w\i , i\o''V„, i κ ',>n I o 0'kuesd' * fer„a'm. h'gy <-,. Precipitation of nn í» complexes should take place. The preeipisátumo; they are collected by centrifugation, (must for example 40öö xg. i pere punés /VG Ssípharosc.! or using a magnet I ipmet A/C magnetic quick rootj, remove it when it becomes flaky, eh pearls? in equal-sized flasks, repeat the collection of the pneciphate and wash with glass, e.g. Fór analysis on o'GSd-'Vll,o b.oumg. os ydVR measuretnenis add 30 ul Iysó. bnlfer and sínre at 4 V,

ArtilRGIlkMdáia:

A typical teaccns kü includes the following1. A positive day! phage solution ?0ö ÖOI) in unit/ul concentration.

2. coffees. negative control Iag solution at a concentration of I per unit/nl.

.!, 96 teakeios. QFCL essay 50 ni final bgat for the broad de-ect.

>k 9s; tc.iki. iö\ íapl 'K iOz/rside 5(i ti! -zs's terminal teeth

5, 24 antibodies sufficient for C'iílP reaction, for which we developed the ISg confeoli system, á. About my IgG stone! 24 for GhlF reaction

?. You have a detailed knowledge of chromatin preparation and examination and the recommended method of a rtisifite.

FUCK! APPLICATIONS

The taiaafofoybun tfo.'existed ken'tnhok advantage vs, (mgy sartahnazfok unod a <e! epnooot. mmn a -mkiemsat as, arn-'s V no, a azt, e. ett a t hl fi r.uk. u' -wik to omaini o.,gxes' hase'doaa they quarrel.

The sFInux' ties<ee\fu<d fall » dig u ndome'» wn x-v fobm vm m λί pia. X'e4.

emek el.fo.e \íO .k nen- V fon aae\.m kimmé η ,-nn is psefop'iK o hanAfo'xsAtrt knnuxut Srsnéi'niO - ^w s ! \o' e > b K t' e > ^! s esi al mt \ . ' sp n.efoe < \ ni d tg. ? 0' nmee ></ íosu.tsi p.ifasnéles works? to the Aoíncvies .The snske basket luk the nnnufooz salo h0zze.5d.tsu alt.·.! . workflows ayosnon köveibeid. The spike is significant; its loss during the work process requires additional labor-intensive and expensive tests, such as narrow-sequencing tests. Can the essays included in the thesis be widely used for the treatment of whites, my salts, for example, miklemic acid circles? proteins, including both histones and non-histone proteins, such as transcription factors, are readily involved in 11-k:onic ex tuses.

Claims (8)

1. \ tu ν.Ά m m (. Xtr, i'kbA'va k'tuhit> 8 nni μΛΦίΗ 0'u'iviibar k «Xx x Irbesartan, whereby the tracer oligonucleotide content of the viral or viral genetic material is traceable; a viral or virion genome encoding the epitope peptide, and where the epitope peptide is a virus or a cosmic virus, the tet <Met \ >> vi 'is found. the message aun oavtecspnx etxc ohm w m -c. The use of claim 1, wherein the akoatesile positive control is: 3, Claim 1: The virus is viral. The use according to any one of claims 1-3, an amdyina virus or a ions bacteriophage, 5, 1-4. Issnmeiic serum for use according to claims 1 to 5. wherein the irmmmpprempstase reaction is a nucleic acid-opto-lipase reaction. selected from the following: · Direct nnkleiasavrin immunoprecipitation and - Ipilate of complex yeast product containing preferential chromatin immunoprecipitation, §. .femtunpreeiprieids set containing the following; - ulcerative nolexia, which is an antigenic epilope for use in thrombosis, - a positive control moiety comprising a nanoparticulate tmkleic acid and an epitope aordic polypeptide imaointase ane'yKn a nvlmnsa, followed by ⁵ tg «mkkx-tmt-reemens» mtaltnaz, and preferably a positive conrio.il bacteriophage. in that particular case a negative control nanoparticle, which judges the octotype and the epitope, but is preferably eluted with text and polypeptides, and which is preferably a &quot; Aan negative control hippo &lt; / RTI &gt; which contains the oligonucleotide and epitope. means for detecting the binding of an antigenic epitope to an antigenic epitope, preferably means for powdering; o control for bacteriophage amplification and, if appropriate, for detection of negative kcmtroll bsteiophage, - a carrier for binding a molecule recognizing a vehicle. preferably beads, from the skeleton the bead is as defined in claim 1, a dermal virus or a viron. 7. The kit for use according to claim 6 for use in moleic acid amino acids, which is a bacteriophage of the virus or vtrion, and which has been used to amplify printers as a means for amplifying them. and / or QPCR primers for amplification of the bacteriophage sequence. I092ő.i6? ®l / $ G> 8, ummuapree spr-aeing procedure, which includes the steps at the foot: - a group of rhinoceroses containing an epitope-containing antigen or target protein, - adding a telisation force molecule that recognizes the epitope of the antigen from a tantigen epitope from one of its sample groups to one or more samples. - a poritic control is provided for an immature particle which contains a polypeptide carrying the epitope of the safflower nucleic acid, wherein the content of oligonucleolide in the tartaric acid can be monitored by oligo; - optionally adding a negative control aa particle containing no epitope from the group of samples to one or more samples, - adding fexinerone oligids to the group of samples - we'll do the mmmtgs'oeápháeíes raids, - wherein the meta particle of claim 1 is demiliacal or in vinyl. The method of claim 8, wherein the immunoprecipitator is an immunoprecipitator mkkylacetic acid complex, preferably a chromatographic mammalian protein complex, which method further comprises the step of inhibiting the binding of the amino acid and the protein, \ {. \ <ion o'ekn Ss \, a \ ' ⁵ Oniövm a leire te you, n eku.u honh' / v i i bound to yiviv material. The h '-ogy u. The method of claims 1 to 4, wherein the detection of binding of the molecule to the epitope shown is carried out as follows: - a region of the mkn · mknissekmkmk which is coded into the spiele represented, and / or - antigen-bound mskieins & v't'mgíncnsekeÉ a chipb.e? wherein at least one of them is complementary to the acleic acid sequence, preferably? regions of the ephope with a coding sequence, and or - shall we sequester the? antigen-bound enclavinmfmgmeest, II. The use of a plurality of traceable oligonucleotide segmented virions encoding eptypppepid in their genome as a control in an immunnapectomy reaction, wherein the epitope peptide is vsrionic bentmatic and is expressed in the form of mpmpreoiptate. preferably 7 or 8 amino acids in length, and capable of binding a Tclamer molecule bound to an epitope carrier, wherein the protein of the carrier protein is first, pr'trmG s v.rgv ezekteameh kmnttsom oh myamma