CN101144821A - Novel technical method for DNA and protein interaction research - Google Patents
Novel technical method for DNA and protein interaction research Download PDFInfo
- Publication number
- CN101144821A CN101144821A CNA2007100555556A CN200710055555A CN101144821A CN 101144821 A CN101144821 A CN 101144821A CN A2007100555556 A CNA2007100555556 A CN A2007100555556A CN 200710055555 A CN200710055555 A CN 200710055555A CN 101144821 A CN101144821 A CN 101144821A
- Authority
- CN
- China
- Prior art keywords
- dna
- albumen
- probe
- protein
- electrophoresis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The present invention relates to a new technical proposal for testing, diagnosing, and studying the biology and the medicine. The present invention is characterized in that the cell protein extract combines into compound earlier than the corresponding antibody molecule, and combines with the null probe into a triple compound of the antibody-albumen-null probe, which can be moved into the non-metamorphic polyacrylamide gel for electrophoresis, and finally an ECL method is used for the irradiance test in darkroom. Compared with the traditional method, the technology has simple and reliable operation without radionuclide element pollution.
Description
Technical field
The present invention relates to the molecular biology research field, be meant especially at DNA in conjunction with albumen and the innovation of associated biomolecule probe repercussion study experimental technique.
Background technology
In the vital movement process of cell, duplicating of DNA and transcribing and modifying of reorganization, RNA, and the infection of virus and propagation or the like all relate to specific DNA section and the interaction between the specific proteins binding factor.Therefore, people just pay close attention to the protein-bonded research of DNA always for a long time.Especially since the recombinant DNA technology development, be separated to the gene that has biological significance in a large number in succession, under this background supported, people began to explore to disclose the external environment factor and grow signal be how to influence or this class problem of transcriptional activity of controlling gene.Gel retardation assay (gel retardation assay) is one of important experimental technique that is used for researching DNA or RNA and protein interaction.In the gel experiment, traditional method is (to claim dna probe again, probe) then with cell protein extract incubation, so just formed the DNA protein complex with radioactive isotope or the plain mark of other biological dna fragmentation to be detected.It is moved in the poly-propionamide gel of non-sex change, and control protein and probe keep bonding state, electrophoresis.Manifest DNA band position by radioactive automatic developing technology (or other proper technology) at last.If there is not the protein that combines with probe in the cell protein, so, all DNA bands all will be concentrated and appear at the gel bottom; Otherwise, will form the DNA-protein complex, because the cause of gel blocking, its distinctive probe-protein complex band is just leaning on the position, back with delayed fashion.Gel retardation assay can be used for not only identifying that whether exist can be with the protein molecule (such as special transcription factor etc.) of a certain specific DNA fragments combination in the specific type cell extract, be used for studying the specificity of the dna sequence dna of this kind combination, but also can be used for catching under specified conditions are intervened the variation of both sides' combination and extraneous biological informations such as influence of intervening to its variation.
But, usually make experimentation become more loaded down with trivial details in traditional testing program, simultaneously, also have to a certain degree radionuclide contamination and injury because dna probe needs mark.The present invention proposes a kind of new solution, its order ground is the above-mentioned defective that will overcome classic method.
Summary of the invention
In order to remedy the above-mentioned deficiency of conventional art, the present invention proposes following technical scheme: cell protein extract elder generation and corresponding antibodies molecule are combined into compound, combine with naked probe (null probe) more afterwards, form antibody-albumen-naked probe three compounds, again it is moved in the poly-propionamide gel of non-sex change, electrophoresis is used ECL method luminous detection in the darkroom at last.
Though the present invention program does not use radioactive nuclide or other biotinylated probe, capture the necessary biological information of research order ground differential protein equally in conjunction with situation, reached experiment purpose.Because the adding of antibody molecule makes the molecular weight of protein-probe bigeminy compound strengthen, behind the electrophoresis, the band that forms in the poly-propionamide of non-sex change lags behind more, thus the outstanding more specificity that has manifested the related protein combination.This method can be used to identify agnoprotein equally, and the band that does not have obvious hysteresis as adding albumen occurs, and this albumen and irrelevant antibody are described, otherwise, as the same.The inventive method has important practical usage to medical diagnosis on disease and biomedical sector research.
Technical solution of the present invention realizes by following technical operation.
1. reagent preparation
(1) Tris-SDS-glycocoll electrophoretic buffer
Tris base 3.03g
SDS 1g
Glycocoll 18.77g
Add ddH
2O is settled to 1000ml.
(2) Tris-SDS-glycocoll-methyl alcohol transfering buffering liquid
Tris base 5.8g
SDS 0.37g
Glycocoll 2.98g
Methyl alcohol 200ml
Use ddH earlier
2O dissolves glycocoll, and Tris base and SDS add 200ml methyl alcohol then, are settled to 1000ml at last.
(3)5×Banding Buffer
20% glycerine
5mM MgCl
2
2.5mM EDTA
2.5mM DTT
250.0mM NaCl
50mM Tris-HCL
0.25vg/vl Poly DI-DC
(4) TBS preparation
1mol/L Tris-HCL ph7.5 10ml
NaCL 8.8g
Use ddH
2O is settled to 1000ml, and when joining TBST, every 100ml TBS adds 4 ℃ of preservations behind 20% the Tween20 mixing of 0.24ml.
(5)Stripping bufer
3-mercaptoethanol 700vl
Tris(PH6.8) 12.5ml
10％SDS 20ml
Deionized water 66.8ml
Cumulative volume 100ml
Wash membrane process: 50 ℃ of shaking table 30min.PBS washes 5min.Using ddH
2O washes film 5min.
Note before washing film: will in TBST, soak 30min at least with the film of crossing.It is gentle that shaking table speed is wanted.
2. electrophoresis
(1) encapsulating 10% separation gel
ddH2O 4.0ml
30％Acr-Bis 3.3ml
pH8.8Tris-HCL 2.5ml
10％SDS 0.1ml
10％AP 0.1ml
TEMED 0.01ml
Polymerization 50min or 4 ℃ spend the night under the room temperature, the glue polymerization well the back under electrophoresis buffering (Tris-SDS-glycocoll) condition at night with 200V voltage prerunning 30min.
(2) go up sample system 15vl
Nucleoprotein: DNA=10: 1
5 * binding buffer liquid 3vl
Nucleoprotein 40vg
DNA 4vg
PolyDI-DC 1vl
ddH
2O Xvl
Under 37 degree conditions, foster 20-30min.Hatch the SDS sample-loading buffer of back adding 3vl, leakage of electricity swimming under the 200v condition.Treating that destination protein runs stopped electrophoresis to 2/3 o'clock of glue.
3. change film and antibodies
3.1 change film with transfering buffering liquid (Tris-SDS-glycocoll-methyl alcohol), it is preceding with glue to change film.Filter paper and ready film (all will be cut into equal size) soak 5min in transfering buffering liquid.
3.2 changeing the film condition is 100mA, 1.5h.Change the film heat production, note in icehouse, carrying out effect more.
3.35% skimmed milk power sealing is spent the night, in 4 ℃.
3.4 certain proportion dilution one is anti-, hatches 1h under the room temperature, washes 3 times with TBST, each 5min washes 1 time time 5min at last with TBS.Dye two of proper proportion dilution then and resist, incubated at room 50min washes 3 times with TBST, and each 5min washes 1 time time 5min at last with TBS.Adopt ECL method luminous detection in the darkroom then.If think that the background height can wash film with Strippingbuffer, antibody is dyed in sealing again, and effect more.
1. materials and methods
11 instruments and reagent
1.1.1 cell line: the strain of MCF7 human breast cancer cell is obtained by the fundamental immunity laboratory.
1.1.2 irradiation instrument: the fixed X ray deep therpy apparatus of homemade X.S.S.205 (FZ) type.
1.13 reagent preparation
(1) Tris-SDS-glycocoll electrophoretic buffer
(2) Tris-SDS-glycocoll-methyl alcohol transfering buffering liquid
(3)5×Banding Buffer(Glycerol，MgCl
2，EDTA，DTT，NaCl，Tris-HCL，Poly DI-DC)
1.2 experimental section
1.2.1 obtaining of target DNA fragment
PCR primer according to p53tumor suppressormRNA sequences Design p53 gene is (synthetic by couple stars biotech firm: lOD, OPC level.) upstream primer: Sense:5 '-AAGGATCCTCTAGAGCCACCGTCCAG-3 ' downstream primer: Antisense:5 '-GGGAATTCCTCCTCCATGGCAGTGAC-3 ' utilizes pcr amplification purpose fragment.Be connected into the order-checking of taking away behind the PCDNA3.1 carrier.The correct back of order-checking transforms, big upgrading grain.Restriction enzyme digestion and electrophoresis then.Cut glue and reclaim the purpose fragment, quantitatively standby.
1.2.2 the extraction of karyon albumen
(1) behind the 4Gy x-ray bombardment MCF7 cell, respectively at 2h, 4h, 8h, 16h, 24h, 48h.Collect cell, extract karyon albumen with triumphant basic endochylema karyon protein extraction kit.
(2) various dose x-ray bombardment MCF7 cell was collected cell after 4 hours, extracted karyon albumen with triumphant basic endochylema karyon protein extraction kit.
(3) Coomassie brilliant blue method protein quantification.-70 ℃ of albumen preservations are standby.
1.2.4 electrophoresis
Encapsulating 10% separation gel (ddH2O4ml, 30%Acr-Bis3.3ml, pH8.8Tris-HCL2.5ml, 10%SDS0.1ml, 10%AP0.1ml, TEMED0.01ml) polymerization under the room temperature, the time is long as far as possible.The glue polymerization well the back under electrophoretic buffer (Tris-SDS-glycocoll) condition with 200V voltage prerunning 20-30min.Last sample system 15vl: nucleoprotein: DNA=10: 1 (5 * binding buffer liquid 3vl, nucleoprotein 40vg, albumen 4vg, PolyDI-DC1vl, ddH
2O X vl).Under 37 degree conditions, foster 20-30min.Hatch the back and add an amount of sample-loading buffer, leakage of electricity swimming under the 200v condition.Treating that destination protein runs stopped electrophoresis to 2/3 o'clock of glue.
1.2.5 change film and antibodies
Change film with transfering buffering liquid (Tris-SDS-glycocoll-methyl alcohol), condition is 100mA, 1.5h.The sealing of 5% skimmed milk power is spent the night, and certain proportion dilution one is anti-and two anti-, adopts ECL method luminous detection in the darkroom then.
2. experimental result
2.1X the time-histories of the dna binding activity of Nucleolin albumen changes behind the radiation exposure MCF7 cell
After adopting albumen hysteresis experimental technique to detect 4GyX radiation exposure MCF7 cell, nucleolin albumen changes with the time-histories of P53 gene 5 ' end non-translational region dna fragmentation in conjunction with activity in the nuclear, accompanying drawing 1. is that the time-histories of the dna binding activity of Nucleolin albumen behind the x-ray bombardment MCF7 cell of intensity 4Gy changes the electrophoresis result synoptic diagram, accompanying drawing 2. is the linear synoptic diagram of the time-histories result of variations of the dna binding activity of Nucleolin albumen behind the x-ray bombardment MCF7 cell of intensity 4Gy, from accompanying drawing 1., compare 2h with false according to group (C group in the accompanying drawing 1.) behind table 1 and the accompanying drawing 2. visible 4GyX radiation exposures, 4h, 8h and 16h descend in conjunction with activity, and 4h reduces the control of p53 in conjunction with the reminding nucleolin of activity.
The time-histories of the dna binding activity of Nucleolin albumen changes behind the table 1.4GyX radiation exposure MCF7 cell
| Time (h) | Grouping | ||||||
| |
2 | 4 | 8 | 16 | 24 | 48 | |
| In conjunction with not combination combination/not combination | 2.530.763.33 | 2.470.852.91 | 2.590.912.85 | 1.070.811.32 | 1.890.932.03 | 1.760.971.81 | 1.981.011.96 |
2.2 the dose-effect of the dna binding activity of Nucleolin albumen changes behind the irradiation MCF7 cell
The interior nucleolin albumen of nuclear changes with the dose-effect of p53 gene 5' end non-translational region dna fragmentation in conjunction with activity after adopting albumen hysteresis experimental technique to detect various dose x-ray bombardment MCF7 cell 4h, accompanying drawing 3. is the electrophoresis result synoptic diagram that behind the time 4h varying strength x-ray bombardment MCF7 cell dose-effect of the dna binding activity of Nucleolin albumen is changed, accompanying drawing 4. is the bar shaped synoptic diagram that behind the time 4h varying strength x-ray bombardment MCF7 cell dose-effect of the dna binding activity of Nucleolin albumen is changed, from accompanying drawing 3., table 2. shines the back with accompanying drawing 4 visible 4h dose-effect experiment demonstration various dose to be compared along with the dosage increase in conjunction with reduced activity according to organizing with false.Prompting is along with dosage increase nucleolin weakens the control of p53.
The dose-effect of table 2. various dose x-ray irradiation MCF7 cell dna binding activity of Nucleolin albumen after 4 hours changes
| Dosage (Gy) | Grouping | ||||
| False irradiation group | 0.5 | 1 | 2 | 4 | |
| In conjunction with not combination combination/not combination | 2.380.683.50 | 2.260.762.97 | 2.410.852.84 | 2.250.733.08 | 2.450.732.95 |
(ending in full)
Claims (1)
1. one kind is used for the biomedical technical scheme of checking, diagnosing, study, its technical characterictic is earlier cell protein extract and corresponding antibodies molecule to be combined into compound, combine with naked probe (null probe) more afterwards, form antibody-albumen-naked probe three compounds, it is moved in the poly-propionamide gel of non-sex change, electrophoresis is used ECL method luminous detection in the darkroom at last.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100555556A CN101144821A (en) | 2007-04-23 | 2007-04-23 | Novel technical method for DNA and protein interaction research |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100555556A CN101144821A (en) | 2007-04-23 | 2007-04-23 | Novel technical method for DNA and protein interaction research |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101144821A true CN101144821A (en) | 2008-03-19 |
Family
ID=39207450
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007100555556A Pending CN101144821A (en) | 2007-04-23 | 2007-04-23 | Novel technical method for DNA and protein interaction research |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101144821A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112920174A (en) * | 2021-02-02 | 2021-06-08 | 上海交通大学 | Photosensitive compound and preparation method and application thereof |
-
2007
- 2007-04-23 CN CNA2007100555556A patent/CN101144821A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| EIITSU NAKAJIMA ET AL.: "Identification of the protein components of mismatch binding complexes in human cells using a gel-shift assay", 《FEBS LETTERS》 * |
| JACOB W. HODGSON ET AL.: "Site-Specific Recognition of a 70-Base-Pair Element Containing d(GA)n Repeats Mediates bithoraxoid Polycomb Group Response Element-Dependent Silencing", 《MOLECULAR AND CELLULAR BIOLOGY》 * |
| STEPHEN DEMCZUK ET AL.: "Identification and analysis of all components of a gel retardation assay by combination with immunoblotting", 《PROC.NATL.ACAD.SCI.》 * |
| TAKESHI TAMAKI ET AL.: "Characterization of a GC-rich region containing Sp1 binding site(s) as a constitutive responsive element of the α2(I) collagen gene in human fibroblasts", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| W. X. TANG ET AL.: "Electrophoretic Mobility Shift Assay Identifies Vitamin D Binding Protein (Gc-Globulin) in Human, Rat, and Mouse Sera", 《ANALYTICAL BIOCHEMISTRY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112920174A (en) * | 2021-02-02 | 2021-06-08 | 上海交通大学 | Photosensitive compound and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wu et al. | Magnetic-nanosensor-based virus and pathogen detection strategies before and during COVID-19 | |
| Chen et al. | Early detection of SARS-CoV-2 seroconversion in humans with aggregation-induced near-infrared emission nanoparticle-labeled lateral flow immunoassay | |
| CN103323599B (en) | Time resolved immunofluorescence detection kit for rabies virus nucleoprotein, and preparation method thereof | |
| CN103439515B (en) | Method for detecting valence of antibody | |
| CN105384803B (en) | A kind of Schistosoma japonicum recombinant protein SjSAPLP4 and its encoding gene and application | |
| CN102906278A (en) | Assay for jc virus antibodies | |
| CN105759046A (en) | Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof | |
| CN106366196A (en) | EpCAM antibody immunomagnetic bead and preparation method thereof | |
| Duan et al. | Dual-detection fluorescent immunochromatographic assay for quantitative detection of SARS-CoV-2 spike RBD-ACE2 blocking neutralizing antibody | |
| CN104004763B (en) | A kind of aptamer of affine hepatitis C core protein and application thereof | |
| CN102580120B (en) | Targeted magnetic resonance imaging (MRI) contrast agent and preparation method thereof | |
| CN112415205A (en) | Kit for detecting EB virus/HCMV and application thereof | |
| CN112501173A (en) | GPC1DNA aptamer and application thereof | |
| CN105601744A (en) | Recombinant antibody resisting influenza a virus nucleoproteins as well as preparation method and application of recombinant antibody | |
| CN102399753B (en) | Specific monoclonal antibody of tilletia controversa kuhn and immunofluorescent detection method | |
| CN106324254A (en) | Anti-insulin antibody detection kit and detection method thereof | |
| CN101603965A (en) | The kit of ELISA competition law quantitatively measuring PEG modified medicaments | |
| CN106404731A (en) | PCT (Procalcitonin) and CRP (C-Reactive Protein) double-label time resolution fluorescence immunoassay method for simultaneously detecting bacterial meningitis and viral meningitis | |
| CN105548539A (en) | Indirect immunofluorescence detection method for Lassa Virus IgG antibody | |
| CN106191286A (en) | Brucellar detection method, test kit and application thereof | |
| CN101144821A (en) | Novel technical method for DNA and protein interaction research | |
| CN105527433A (en) | Fluorescence method for detecting tumor marker | |
| ES2567091T3 (en) | Amine compounds for the selective preparation of biological samples | |
| CN102183666B (en) | Liquid phase chip for detecting twelve pathogen antibodies in blood serum sample in high flux, and preparation method and using method thereof | |
| CN110274982A (en) | The quantitative detecting method of rabies viruses inactivation antigen |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20080319 |