CN107793468A - A kind of method for detecting DNA damage - Google Patents
A kind of method for detecting DNA damage Download PDFInfo
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- CN107793468A CN107793468A CN201710497018.0A CN201710497018A CN107793468A CN 107793468 A CN107793468 A CN 107793468A CN 201710497018 A CN201710497018 A CN 201710497018A CN 107793468 A CN107793468 A CN 107793468A
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- C07K1/16—Extraction; Separation; Purification by chromatography
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- C—CHEMISTRY; METALLURGY
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention provides a kind of detection method of DNA damage, particularly by detection multicomponent Ku albumen so as to detecting the method for DNA damage, including：Peptide is extracted from cell pyrolysis liquid, multicomponent peptide is obtained by tandem affinity purification；Analyzed using Two way chromatograms post, LC/MS analyzes the peptide；Dump valve controls waste liquid flow velocity so that ESI flow velocitys are in 5nL/min.First dimension chromatographic column is SCX or RP, and the second dimension chromatographic column is RP.The present invention observes to be passed through in the time course after gamma Rays more than 5 hours by cell, the change of the horizontal related complex of silver staining gel, and the dynamic of protein complex under the conditions of these is decoded using iTRAQ stable isotope combination RP RP MS.
Description
Technical field
The present invention relates to a kind of detection method of DNA damage, more particularly to it is a kind of by detect multicomponent Ku albumen so as to
The method for detecting DNA damage.
Background technology
It is a variety of phase interactions by protein, nucleotides and small metabolic molecule that normal and distortion biology is dominant
Caused by driving.Increasing evidence shows that so-called system-level tentative strategy can increase traditional hypothesis driving
Approach, with for describing reaction of the cell in the case where receiving pressure, infection, disease and other biological or environmental disturbances.According to generation
Amount and detailed degree compare, and the data obtained by a large amount of protein properties descriptive studies, generally lag behind DNA and RNA.
As a rule, the technology barrier of accurate large-scale protein matter group research and development is hindered, is due to current substantial amounts of spectroscopic assay
Instrument is caused by detecting and can use the limitation in mechanics scope, therefore, in face of consolidating for production " complete " protein catalog
Have any problem, many researcher's selections are using the amendment after analysing protein subclass or specific translation as goal in research.Largely
Case study evidence show that the biomaterial of concentration be used to concentrate research, but can but be answered across large-scale sample
Polygamy, and the protein and the concentration range of peptide produced, surpass the analysis energy of contemporary a large amount of spectrometric techniques
Power.
By to complex biological sample not between analyze, as a result show that LC-MC technologies still have some analyzing defects,
Although MS/MS service life improves stably, simultaneously in the amino acid quantity of extraction and analysis, can nevertheless suffer from
Restrict.These data concentrated and observation result have expedited the emergence of excessive work, for improving amino acid and protein separation
Chromatogram peak out capacity, reduce the analyte quantity of MS/MS required extractions within the unit interval.Multidimensional fractionation art has become most
Popular method, to be analysed in depth to the complicated ispol derived from bio-extract.
Tandem affinity purification technology (TAP) is the technology that substantial amounts of spectroscopic assay is connected, currently used for describing multicomponent
Protein complex.But current TAP schemes can only based on physiology in terms of relevant condition high yield and spy are provided
The valuable analyze data to obtain is analyzed in the protein expression of the opposite sex, some requirements by efficient and depth LC-MC,
Current scheme still can not solve.
The content of the invention
The invention provides a kind of method for detecting DNA damage, chromatographs platform by multidimensional and is combined with LC/MS,
There is provided and really receive the component of stream (nL/min) degree online.
The method of detection DNA damage provided by the invention, including：
Peptide is extracted from cell pyrolysis liquid, multicomponent peptide is obtained by tandem affinity purification；
Analyzed using Two way chromatograms post, the first dimension chromatographic column is SCX (strong cation exchange chromatography) or RP (reverse phase liquids
Chromatogram), the second dimension chromatographic column is RP；Multicomponent peptide is loaded into the first dimension chromatographic column by automatic sampler, then according to SCX or RP
Selection injection salt or organic elution agent, produce the first dimension component, be acidified with fumaric acid and dilution in acetonitrile；The position valve of 6 hole 2
With the second dimension chromatogram column coupling；Organic elution agent is injected into the second dimension chromatographic column, peptide is eluted；
LC/MS analyzes the peptide；Dump valve controls waste liquid flow velocity so that ESI flow velocitys are in 5nL/min.
In an advantageous embodiment, the multicomponent peptide is the Ku albumen compositions of FLAG-HA marks.
In a kind of more preferred embodiment, the Ku albumen can be in Ku86, Ku70, Ku80 any one or it is several
Kind.
In an advantageous embodiment, the multicomponent peptide is marked with iTRAQ.
In an advantageous embodiment, the tandem affinity purification method includes：The nucleus obtained from cell pyrolysis liquid
It is suspended in lysis buffer, collects supernatant, adding coupling has the spheroid carrier of antiflag antibody, in 4 DEG C of cultures；Will
Gained sphere is cleaned with lysis buffer.
In a kind of more preferred embodiment, the tandem affinity purification method includes：The cell obtained from cell pyrolysis liquid
Core is suspended in lysis buffer, collects supernatant, and adding coupling has the spheroid carrier of antiflag antibody, in 4 DEG C of cultures；
Gained sphere is cleaned with lysis buffer.
In a kind of more preferred embodiment, the carrier can be selected from cellulose, Ago-Gel, sephadex,
Any one or a few in polyacrylamide gel, Bio-Glas, chitosan.
In an advantageous embodiment, the first dimension chromatographic column includes the inlet cover of capillary and lid on the capillary,
According to SCX or RP selection, with strong cation-exchanging resin or chromatographic grade silicones packed bed.
Wherein, the capillary inner diameter is preferably 100 microns.
Wherein, the inlet diameter of the inlet cover is preferably 0.5 micron.
In one preferred embodiment of the invention, it is of the invention by detecting the protein component of Ku albumen compositions
Change, to detect DNA damage.
Ku albumen compositions are played an important role in DNA damage and repair process, and the present invention observes to pass through by cell
Cross in the time course after gamma Rays more than 5 hours, the change of the horizontal related complex of silver staining gel, and profit
The dynamic of protein complex under the conditions of these is decoded with iTRAQ stable isotope combinations RP-RP-MS.
Brief description of the drawings
Fig. 1 is the analysis for the peptide that SCX-RP separates with RP-RP；Figure 1A and B is respectively the quantity for differentiating peptide and amino acid,
Fig. 1 C are histidine detection and identification result；
Fig. 2 is that MS detects peak intensity before STCTGVEMFR peptides；Fig. 2A and B is respectively that SCX and RP first ties up component, Fig. 2 C
Peptide is eluted for the first dimension component in RP-RP and SCX-RP；
Fig. 3 is the Ku86 analysis of protein of the FLAG-HA marks of TAP methods purifying；
Fig. 4 is change of the Ku86 albumen compositions of FLAG-HA marks in DNA damage；
Fig. 5 is that the biochemistry of the Ku86 albumen compositions of the quantitative FLAG-HA marks of iTRAQ PR-RP-MS/MS can
Row.
Embodiment
The structure of chromatographic column
First dimension chromatographic column (RP or SCX) by 360 microns outer diameters, 100 micron inside diameters melting silicon capillary on cover
0.5 micron of inlet cover composition.35 microns of XBridge C18 chromatographic columns silicone grease and SCX mediums are respectively used to fill the first dimension
RP and SCX chromatographic column beds.
Multicomponent peptide is loaded into the first dimension chromatographic column by automatic sampler, then injects salt according to SCX or RP selection or has
Machine eluant, eluent, the first dimension component is produced, with 0.1wt% fumaric acid and the dilution acidifying of 3wt% acetonitrile mixed solutions；The position of 6 hole 2
Valve and the second dimension chromatogram column coupling；Organic elution agent is injected into the second dimension chromatographic column, peptide is eluted.
Cell culture and γ radiation
Ku86 (FLAG-HA marks Ku86) the HeLa S3 cells of affine FLAG and HA peptide epitopes mark are expressed in 15cm
Grown in tissue culture dishes, each culture dish includes 25 milliliters of DMEM, supplements 5% fetal bovine serum.Outside cell mass supplementary quota
Cultivate 3 days, be then further cultured for 24 hours in 37 DEG C, 5.0% carbon dioxide after 3 milliliters of culture mediums.Gammecell 40 is irradiated
Instrument, equipped with two Ce 137 sources, gamma-rays irradiation is carried out to HeLa S3 cell masses, to 12 diameters, 15 centimetres of fusions altogether
Tissue Culture Dish carries out 10Gy irradiations with 1.1Gy/min center close rate.Then cell is put back into incubator, using 4 plates as one
Group, respectively after 30 minutes, 2 hours and 5 hours, remove one group.Under above-mentioned condition, the drop of cell survival rate is not observed
Low or Apoptosis sign.Parallel processing is carried out using 4 plates as one group (control group), does not receive gamma-rays irradiation.
The preparation of nucleus
Cell is transferred in 50 milliliters of conical pipes (2 plate tube).Then, cell is placed in refrigerated centrifuge,
500xg, precipitate 3 minutes at 4 DEG C.Supernatant liquor is removed, cell precipitation is suspended in 50 milliliters at 4 DEG C of 1xPBS solution
In (137mM sodium chloride, 2.7mM potassium chloride, the hypophosphite monohydrate disodium hydrogens of 4.3mM seven), then it is again placed in centrifuge,
500g, precipitate 3 minutes under 4 degrees Celsius.
Cell is suspended in 10 milliliters of 1xPBS, then centrifuged 3 minutes under 4 degrees Celsius, 500g.It is clear to remove upper strata
Liquid, sediment is suspended in in the hypotonic buffer liquid for decupling sediment volume (Tris of 10mM pH values 7.3,10mM chlorinations
Potassium, 1.5mM magnesium chlorides, 10mM beta -mercaptoethanols).Suspension is centrifuged 3 minutes under 4 degrees Celsius, 800g.It is clear to remove upper strata
Liquid, sediment is suspended in 1 milliliter of hypotonic buffer liquid, is transferred to 7 milliliters of Dounce tissue homogenizers.By cell rupture, so
Homogenate lysate is divided equally into two 1.5 milliliters of microcentrifugal tubes afterwards.By cell pyrolysis liquid under 4 degrees Celsius, 1000g from
The heart 4 minutes, obtain nuclear particle and the cytoplasm in supernatant liquor.
After centrifugation, supernatant liquor is separated into 1.5 milliliters of microcentrifugal tubes.Each core precipitation be resuspended in 1 milliliter it is low
Buffer solution is oozed, and is centrifuged 10 minutes under 4 degrees Celsius, 1000g.Remove supernatant liquor, the remaining fast quickly cooling of nucleus liquid nitrogen
Freeze and be stored in subzero 80 degrees Celsius.
The tandem affinity purification of the Ku86 compounds of FLAG-HA marks
Freezing nuclear particle is resuspended in lysis buffer (30mM sodium chloride, 10% glycerine, the 50mM of 2.5 times of particle volumes
PH7.5 trishydroxymethylaminomethane, 0.2%NP-40,1mM EDTA, Roche EDTAfree protease inhibitor cocktails)
In.It is placed on 4 degrees Celsius of rotation platforms 30 minutes.The karyorhexis thing of gained centrifuges 10 points at 4 degrees Celsius with 14000x rpm
Clock.Supernatant liquor is moved in fresh tube, remainder particulate is abandoned.
With the sepharose 4B (80 microlitres) of Sigma M2 mouse antiflag antibody couplings, in 800 microlitres of lysis buffers
Middle cleaning three times, is then resuspended in 80 microlitres of lysis buffers.Suspended beads liquid is added separately to four (0 point of core samples
Clock, 30 minutes, 2 hours and 5 hours) in, sample cell is cultivated 4 hours on 4 degrees Celsius of rotation platforms.
These pearls are cleaned three times with 200 microlitres of lysis buffer, then with 200 microlitres of HA buffer solutions (30mM chlorine
Change sodium, 10% glycerine, 50mM Ph7.5 Tirs, 0.05%NP-40,1mM EDTA, Roche EDTAfree protease inhibitors
Mixture) cleaning is once.
Ku86 protein complexes are added in 18.5 microlitres of HA buffer solutions, and 1.5 microlitres of concentrations are contained in these buffer solutions
FLAG peptides (2mM), then carried out 20 minutes, eluted under 4 degree celsius temperatures with 1200rpm rotating speeds.FLAG is repeated once to wash
It is de-；Eluent is merged, and with 1400rpm at 4 deg. celsius by 0.45 micrometer PVDF membrane (Millipore, block in Bill,
Massachusetts) 30 seconds filtered.
Then, 80 microlitres of the HA sepharose 4Bs being coupled are washed three times in 800 microlitres of HA buffer solutions, then suspended
In 80 microlitres of HA buffer solutions.HA pearls test suspension (25 microlitres) decile is added into each FLAG eluents, then with
1200rpm rotating speeds are stayed overnight at 4 deg. celsius.Each sample with 1200rpm and 4 degree Celsius respectively with 18.5 microlitres of HA buffer solutions
With elution in 1.5 microlitres of concentration HA peptides (2mM) twice.By eluant, eluent filtering as obtained above and with -80 degrees Celsius of preservations.
Prepare the polypeptide of iTRAQ marks
By the eluent of the frozen protein compound at four time points by adding 0.1wt%Rapigest SM reagents
It is denatured and is decomposed with 5mM dithiothreitol (DTT).Solution is cultivated 30 minutes at 60 degrees Celsius.After equilibrating to room temperature, it will be denatured
Protein solution with 750ng be sequenced level trypsase (Promega polyunsaturated fatty acids and multivitamin preparation) 37
Cultivate 24 hours and digested in degree Celsius carbon dioxide.
After digestion, addition 0.5%v/v trifluoroacetic acids are cracked in RapiGest reagents.Then sample is taken the photograph 37
Cultivated 30 minutes in the CO2gas incubator of family name's degree.The reagent is passed through into 4 degrees Celsius of 20 points of centrifugations (rotating speed 14000rpm)
Clock.Remove supernatant liquor, and the desalination on 96 orifice plates of anti-phase elution.Each hole is rinsed with 400 microlitres of 40% acetonitriles/0.1%TFA
Once, then rinsed with two addition 0.1%TFA 400 microlitres of cleaning solutions.Peptide solution is loaded to hole, with 400 microlitres
0.1%TFA is rinsed twice, and is eluted with 50 microlitres of 40% acetonitrile/0.1%TFA for being divided into two parts.
Sample is dried by centrifugal concentrating, and is reconstructed in 14 microlitres of 0.5M triethyl ammonium bicarbonate pH8.5 buffer solutions.
Meanwhile clean about 20 microlitres of dry pearls five times with the ultra-pure deionized water of 1 ml aliquots, for preparing a 40 microlitres work
Property mercaptan Ago-Gel 4B pearls.Then it washed once with 200 microlitres of 0.5M TEAB, and reconstructed with 40 microlitres of 0.5M TEAB.
From remaining pearl solution, 14 microlitres are taken to be added in four peptide samples respectively, and it is 1 small to be vortexed under 1400rpm with room temperature
When.
After centrifugation, supernatant is transferred in fresh tube, abandons bead.In this step, four iTRAQ examinations
117) agent passage (114,115,116 and is separately added into 70 microlitres of alcohol, each channel volume is changed into 90 microlitres.Respectively four
The iTRAQ reagents of 40 microlitres of alcohol and 36 microlitres of reconstruct are added in individual sample, and are carried out as follows：114-0 minutes,
115-30 minutes, 116-120 minutes, 117-300 minutes.Resulting solution is cultivated to a hour at room temperature.
Sample volume is concentrated to 30 microlitres respectively by centrifuge separation, it is further dilute to add 100 microlitres of 0.1%TFA
Release.Four samples are combined, and desalination in anti-phase micro- elution disk is placed according to being previously described.It will be combined with centrifuge
Sample is dried, and is added 65 microlitres of 20mM pH10 ammonium formates and is reconfigured.Sample is divided into 10 microlitres of portions, equivalent to
The initial cell material of two disk components, is then stored in -70 degrees Celsius of LC-MS.
Reference picture 1, the present invention analyze the first dimension (SCX and RP) peak value, Fig. 1 cover 10,20,30,40 in first
The experiment of dimension component, compared to SCX-RP, PR-RP provides the exclusive polypeptide (Figure 1A) and protein (figure of higher amount
1B) identify, it was further observed that RP-RPP can provide the identification (Fig. 1 C) of more polypeptides from each protein.Pass through respectively
In the 16 and fifteen amino acid remnants that SCX-RP and RP-RP are obtained, analyzed in all conditions, the polypeptide of identification is averaged
Length is still very consistent.Fig. 1 c show that only histidine is enriched with polypeptide under by SCX-RP, and is carried out by RP-RP
The amino acid of polypeptide identification, which is not observed statistically, notable deviation.
Due to MS/MS random natures, verify that given peptide whether there is in continuous first dimension point by peptide identification
It will become under evaporating sufficiently complex.In order to make great efforts to mitigate this effect, we use precursor mass tolerance (± 1Da) and the second dimension
The combination of LC elution times (± 1 minute) is spent, it is neighbouring each in initial identification occurs to explore in the fractionation of the first dimension
Kind peptide.Fig. 2A and 2B provides the representative data of peptide, these data by STCTFVEMFR by various technologies 10,20,
30th, identified under 40 the first dimensions.7 the first dimension groups spanned by the SCX-RP presumption more propeptides of match cognization
Point, but only 1 component elution in RP-RP analyses.Therefore, compared to SCX-RP, RP-RP, STCTGVEMFR are passed through
It was observed that precursor signal intensity maximum it is considerably higher.Generally speaking it is averaged by RP-RP what the first dimension component obtained
Numerical value spans the polypeptide of all identifications, it was demonstrated that RP-RP shows excellent peak strength (Fig. 2 C) all the time.
Fig. 3 gives the more propeptide intensity detected jointly and knot is individually identified respectively and by SCX-RP and RP-RP
The block diagram of fruit contrast.There is overlapping (Fig. 3 A) in distribution separately through the SCX-RP polypeptides identified and the polypeptide identified jointly,
And separately through the polypeptide that RP-RP is identified compared to the polypeptide identified jointly by two kinds of technologies, gather to relatively low rich
Spend scope (Fig. 3 B).Integrated data shows that RP-RP is in the total and dynamic range that peptide identifies, than SCX-RP more preferably.
The present invention carries out the Ku86 that core fractionation extracts FLAG-HA marks to FLAH-HA Ku HeLaS3 cell lines, and
Tandem affinity purification experiment is carried out to it.Parallel tandem affinity purification experiment is carried out to the HeLaS3 cell masses of paternal line simultaneously,
With as a control group.Dissolved with SDS-PAGE and shown with Silver stain.It is very huge to form the protein amounts of Ku compounds,
These protein cover extensive stoichiometry and molecular weight (Fig. 4 A).Protein is not detected in control group, this shows
Most protein band is decomposed by the SDS-PAGE that specified protein interacts in Ku compounds is represented.
LC-MA/MS replicate analysis are carried out for SCX-RP and RP-RP, as shown in figure 4, RP-RP is able to detect that ratio
Specially peptide and protein group significantly more SCX-RP；About 94% is limited in all polypeptides detected by RP-RP
In the first single dimension component (Fig. 3 D), and corresponding SCX-RP experiments, only about 83% identification polypeptide can reach
This standard.In subsequent RP-RP experiments, by Ku compound deciles, and pass through first dimension of 20,30 and 40 times respectively
Component obtains thing and analyzed.Fig. 4 B and 4C show the sustainable growth of peptide and protein identification quantity, and this is the work of depth component
With higher peak strength can be obtained by RP-RP fractionation by demonstrating again that.
In these experiments, it was observed that many be previously accredited as the protein for forming Ku 70/86, including adult progeria
Poly- (ADP- ribose) polymerase I (PARP-1), the YY1 of GAP-associated protein GAP (WRN), RNA dependence unwindase A/ cores DNA are relied on
Unwindase II/ (RHA/NDHII/DDX9), and several heterogeneous nucleoprotein (hnRNPs).Generally speaking, the present invention dramatically increases
The catalogues of Ku albumen composition compositions.
Comparison (Fig. 5 A) display of gel lane, the change of the protein component of Ku compounds is the most under these conditions
It is delicate.Each TAP eluents remainder is dissolved with trypsase, marked with iTRAQ reagents, then with 40 grades of RP-RP MS/
MS is analyzed.0,0.5 hour, 2 hours and 5 hours after gamma-rays processing, the present invention examines altogether in Ku albumen compositions
Survey and quantified 321 kinds of protein.It is observed that several known protein that can be interacted with Ku, including DNA-
PKcs, WRN, NCL and H2AX.Contrast the result specifically tested and analysis (Fig. 4,40 RP-RP fractionation) phase of base compounds
Than the overlapping part of protein identification is up to 82%, illustrates TAP and RP-RP components (Fig. 5 B) repeatability again.Fig. 5 C
Protein component can generally reduce the result of DNA damage in display compound.
With being quantified based on iTRAQ during these researchs, the MS scannings in foregoing each MS/MS projects
Peptide identification is generated, and records other and appears in the quantity that m/z scopes -0.5 in peptide identification arrive+0.1Da peptide precursor.When
When precursor intensity exceedes peptide 20% relatively interested, co-elute species are labeled as potential interference thing.By this relatively crude
Shallow criterion, it is observed that in polypeptide identification caused by potential pollutant and 10 fractionation SCX-RP experiments
12.7% is relevant.In 10 fractionation RP-RP analyses of control, only 7.4% polypeptide identification co-elute thing may interfere with thing
Kind.With it is observed that the results that are improved to peak strength of RP-RP it is consistent, potential precursor pollutant ratio is most advanced
2.4% is reduced under degree component.
The specific embodiment of the present invention is described in detail above, but it is intended only as example, it is of the invention and unlimited
It is formed on particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and
Substitute also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and
Modification, all should be contained within the scope of the invention.
Claims (10)
- A kind of 1. method for detecting DNA damage, it is characterised in that step includes：Peptide is extracted from cell pyrolysis liquid, multicomponent peptide is obtained by tandem affinity purification；Analyzed using Two way chromatograms post, the first dimension chromatographic column is SCX or RP, and the second dimension chromatographic column is RP；Automatic sampler will be more Component peptide is loaded into the first dimension chromatographic column, then injects salt or organic elution agent according to SCX or RP selection, produces the first dimension group Point, with fumaric acid and acrylonitrile dilution acidifying；The position valve of 6 hole 2 and the second dimension chromatogram column coupling；Noted into the second dimension chromatographic column Enter organic elution agent, peptide is eluted；LC/MS analyzes the peptide；Dump valve controls waste liquid flow velocity so that ESI flow velocitys are in 5nL/min.
- 2. according to the method for claim 1, it is characterised in that the multicomponent peptide is that the Ku albumen of FLAG-HA marks is answered Compound.
- 3. according to the method for claim 1, it is characterised in that the multicomponent peptide is marked with iTRAQ.
- 4. according to the method for claim 1, it is characterised in that the tandem affinity purification method includes：The nucleus obtained from cell pyrolysis liquid is suspended in lysis buffer, collects supernatant, and adding coupling has antiflag The spheroid carrier of antibody, in 4 DEG C of cultures；Gained sphere is cleaned with lysis buffer.
- 5. according to the method for claim 4, it is characterised in that the lysis buffer includes：30mM sodium chloride, 10wt% Glycerine, 50mM pH value are 7.5 Tris, 0.05wt%NP-40,1mM EDTA, and Roche mixes without EDTA protease inhibitors Thing.
- 6. according to the method for claim 4, it is characterised in that the carrier is selected from cellulose, Ago-Gel, glucan Any one or a few in gel, polyacrylamide gel, Bio-Glas, chitosan.
- 7. according to the method for claim 1, it is characterised in that the first dimension chromatographic column includes capillary and covered in capillary Inlet cover on pipe, according to SCX or RP selection, with strong cation-exchanging resin or chromatographic grade silicones packed bed.
- 8. according to the method for claim 7, it is characterised in that the capillary inner diameter is 100 microns.
- 9. according to the method for claim 7, it is characterised in that the inlet diameter of the inlet cover is 0.5 micron.
- 10. according to the method for claim 1, it is characterised in that by the protein component for detecting Ku albumen compositions Change, to detect DNA damage.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101528925A (en) * | 2006-09-21 | 2009-09-09 | 法国居里学院 | Dbait and uses thereof |
| CN101622351A (en) * | 2007-01-12 | 2010-01-06 | 法国国家科学研究中心 | Dbait and independent purposes thereof |
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2017
- 2017-06-26 CN CN201710497018.0A patent/CN107793468A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101528925A (en) * | 2006-09-21 | 2009-09-09 | 法国居里学院 | Dbait and uses thereof |
| CN101622351A (en) * | 2007-01-12 | 2010-01-06 | 法国国家科学研究中心 | Dbait and independent purposes thereof |
| US20100022622A1 (en) * | 2007-01-12 | 2010-01-28 | Marie Dutreix | Dbait and its Standalone Uses Thereof |
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| Title |
|---|
| AIDAN J DOHERTY等: "Identification of bacterial homologues of the Ku DNA repair proteins.", 《FEBS LETTERS》 * |
| FENG ZHOU等: "Online Nanoflow RP-RP-MS Reveals Dynamics of Multicomponent Ku Complex in Response to DNA Damage", 《JOURNAL OF PROTEOME RESEARCH》 * |
| HENRY C.H.LAW等: "A versatile reversed phase-strong cation exchange-reversed phase (RP–SCX–RP) multidimensional liquid chromatography platform for qualitative and quantitative shotgun proteomics.", 《ROYAL SOCIETY OF CHEMISTRY》 * |
| 占大钱等: "Ku蛋白与肿瘤", 《世界华人消化杂志》 * |
| 李晶晶等: "Ku蛋白在肿瘤DNA损伤修复中作用", 《中华实用诊断与治疗杂志》 * |
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