CN101498734A - Method for simultaneously turning two films and marking protein TRX and procaspase12 - Google Patents
Method for simultaneously turning two films and marking protein TRX and procaspase12 Download PDFInfo
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- CN101498734A CN101498734A CNA2009100941697A CN200910094169A CN101498734A CN 101498734 A CN101498734 A CN 101498734A CN A2009100941697 A CNA2009100941697 A CN A2009100941697A CN 200910094169 A CN200910094169 A CN 200910094169A CN 101498734 A CN101498734 A CN 101498734A
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Abstract
The invention relates to a method for simultaneously rotating two films and marking two proteins, namely TRX and procaspase12. The method includes the steps of total protein extraction of cells, protein quantifying, gel electrophoresis, protein blotting, simultaneously marking of antibodies, X-ray film developing, and the like. The invention aims at building up a method which is characterized in that two PVDF films are used for simultaneously marking and detecting the two target proteins by using different antibodies after SDS-PAGE and protein blotting are carried out on the total protein in cells, meanwhile, the method is compared with the traditional method which has the steps of stripping films and then marking antibodies, and the accuracy, the reproducibility, the stability, and the like of the method are evaluated. The invention is simple and easy to carry out, can well represent the test result, and provides a method which is simpler and easier and saves time and samples, thereby having actual application value.
Description
Technical field
The invention belongs to biology field, relate to and change two films simultaneously, the method for labelled protein TRX and procaspase 12, thus detect the situation of change of two kinds of destination proteins.
Background technology
Protein immunoblot (Western Blot) is according to the specificity of the antigen-antibody method in conjunction with certain albumen in the detection of complex sample.
Western blot is a kind of new immune biochemical technology that grows up on gel electrophoresis and solid-phase immunoassay technical foundation, mainly comprises gel electrophoresis, Western blot (commentaries on classics film) and immunology detection three parts.
Gel electrophoresis is that the protein in the testing sample is divided into band according to the molecular weight size in gel.Western blot is meant to be transferred to the protein example of gel electrophoresis separation on the solid phase carrier (for example cellulose nitrate film or pvdf membrane), solid phase carrier is with non-covalent bond form adsorbed proteins, and can keep the polypeptide type and the biologic activity thereof of electrophoretic separation constant.As antigen, play immune response with the protein on the solid phase carrier or polypeptide, react with ELIAS secondary antibody again, through substrate colour developing or radioautograph protein ingredient with the specific destination gene expression that detects electrophoretic separation with corresponding antibody.
At present, gel electrophoresis technology has been applied to detect the expression of protein level.But do not see in the prior art to have and change two films simultaneously, the report of the method for labelled protein TRX and procaspase 12.
Summary of the invention:
The present invention is intended to by setting up total protein in a kind of cell through behind SDS-PAGE and the Western blot, with two kinds of methods that destination protein detects with tense marker simultaneously with different antibodies on two pvdf membranes.And with this method and traditional stripping again the method for labelled antibody compare, to the accuracy of this method, aspects such as repeatability and stability are estimated, for a kind of simple method that provides is provided multiple protein, promptly save time and save sample, using value is arranged.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
Once change two films, the method for labelled protein TRX and procaspase 12 simultaneously comprises extraction, protein quantification, gel electrophoresis, the Western blot of total protein of cell, the same tense marker and the X-ray film developing step of antibody.
Particularly, extract total protein of cell, behind protein quantification, carry out gel electrophoresis; Albumen with two overlapping being placed on the glue of onesize pvdf membrane, is carried out mark so that distinguish after gel electrophoresis, change the film condition and be: U=100V, I=350mA, T=70min; Antibody with two films difference mark TRX and procaspase 12 carries out immunology detection, X-ray film developing testing result.
Wherein, the dilution ratio of two kinds of antibody is respectively: TRX1:5000, procaspase12 1:800.
More specifically, extract the PC12 total protein of cell, utilize BCA method pair cell total protein quantitative; The total protein that extracts in the cell is carried out gel electrophoresis; With wet commentaries on classics method the albumen on the glue is transferred on the pvdf membrane; One anti-mark, antigen-antibody is washed film with TPBS after hatching 70min; Two anti-marks are washed film with TPBS after hatching 70min with horseradish peroxidase two is anti-with antigen antibody complex; Add chemical luminous substrate and develop, obtain the band of two kinds of destination proteins; Check the repeated and stable of this method by traditional stripping method.
Change the foundation of two kinds of antibody methods of two membrane markers in the protein immunoblot of the present invention simultaneously, content comprises following step:
A. the present invention is an example with the PC12 cell, extracts the PC12 total protein of cell, utilizes BCA method pair cell total protein quantitative.
B. the total protein that extracts in the cell is carried out gel electrophoresis.
C. with wet commentaries on classics method the albumen on the glue is transferred on two pvdf membranes.
D. an anti-mark, antigen-antibody is washed film with TPBS after hatching 70min.
E. two anti-marks are washed film with TPBS after hatching 70min with horseradish peroxidase two is anti-with antigen antibody complex.
F. add chemical luminous substrate and develop, can obtain the band of two kinds of destination proteins on two films.
G. detect with traditional stripping method and compare, the repeatability of this technology and stable.
Description of drawings:
Fig. 1 is that a glue changes the purpose band that two films detect TRX and procaspase 12 simultaneously;
Fig. 2 is for traditional stripping, TRX that labelled antibody obtains and the developing result of procaspase 12 again.
Embodiment:
Below in conjunction with embodiment, further set forth the present invention, but do not limit the present invention with this.
Embodiment 1:
Glue changes two pvdf membranes, with two kinds of antibody of tense marker:
(1) extracts total protein of cell: discard nutrient culture media behind the medicine irritation PC12 cell 24h, add the residual nutrient culture media of 2ml left and right sides PBS flushing cell surface, discard washing lotion.Add and stop digestion about 0.25% trypsinization 5min, the centrifugal 5min of 1500rpm, discard supernatant, add the 1mlPBS suspension cell, 1500rpm4 ℃ of centrifugal 5min, discard supernatant, add 100 μ l cell pyrolysis liquids, about cracking 1h, 4 ℃ of 15000rpm are centrifugal 15 minutes on ice, supernatant (total protein of cell) is transferred in the new dorf pipe-80 ℃ of preservations.
(2) quantification of protein: press the operation of BCA quantification of protein kit operation instructions, working sample concentration.
(3) electrophoresis: the total protein of cell that extracts is carried out the discontinuous gel electrophoresis of denaturing polyacrylamide (SDS-PAGE) after 95 ℃ of thermal denaturations.15% separation gel 10ml: sterilized water 2.3ml, 30% polyacrylamide 5.0ml, Tris (PH=8.8,1.5M) 2.5ml, 10% SDS 0.1ml, 10% APS 0.1ml, TEMED 4 μ l.Compression glue 4ml: sterilized water 2.7ml, 30% polyacrylamide 0.67ml, Tris (PH=6.8,1.0M) 0.5ml, 10% SDS, 40 μ l, 10% APS, 40 μ l, TEMED 4 μ l.With ready sample liquid with dye albumen marker in advance and go up sample respectively, electrophoretic separation albumen.Deposition condition: U=180V, I=100mA, T=40min.After finishing, changes electrophoresis film.
(4) change film: owing to all be with negative charge through the albumen on the glue of electrophoresis, and be that film is changeed in constant current, therefore the albumen that goes on the film is uniform, and go on first film with second film on albumen all be the same with the ratio of total protein, so change film according to the order of negative pole-cotton-three metafiltration paper-glue-two pvdf membrane-three metafiltration paper-cotton-positive pole, change the film condition: U=100V, I=350mA, T=70min.Carry out in the frozen water.
(5) sealing: two pvdf membranes that will take a turn for the better spend the night with the sealing of 5% skimmed milk, are that second day antigen-antibody mark is prepared.
(6) one anti-marks: two pvdf membranes that sealing is spent the night take out from 5% skimmed milk, clean three 5min with TPBS, mark one is anti-, two pvdf membranes are different one anti-of mark respectively, the present invention is an example with TRX and procaspase 12, TRX 1:5000 dilution, procaspase 12 1:800 dilution.One anti-mark 70min.
(7) two anti-marks: after an anti-mark is finished,, begin two anti-marks with give a baby a bath on the third day after its birth time 5min of TPBS.Two of TRX of the present invention and procaspase 12 anti-is the goat-anti rabbit, and extension rate is 1:5000.Two anti-mark 70min.
(8) X-ray film developing: after two anti-marks were finished, with give a baby a bath on the third day after its birth time 10min of TPBS, three 5min added millipore chemical luminous substrate (1ml/10cm
2), in the darkroom, develop.
Embodiment 2:
Detect the accuracy and the stability of the method for embodiment:
Utilize traditional stripping method to carry out the mark of two kinds of different antibodies, thereby detect accuracy of the present invention.
Concrete grammar is as follows:
(1) mark that will on the X-ray sheet, develop the pvdf membrane of TRX antibody add 10ml stripper solution (beta-mercaptoethanol 0.27ml, 10% SDS 2ml, 1.0M Tris 6.8 625 μ l), hatch 30min for 60 ℃, clean three 10min with TBPS, put into 4 ℃ of sealings of 5% skimmed milk then and spend the night.
(2) film is taken out from skimmed milk, clean three 10min with TPBS, mark procaspase 12 1 is anti-, hatches 70min.
After (3) one anti-marks are intact, clean three 5min with TPBS, mark two is anti-, hatches 70min.
After (4) two anti-marks are finished, clean three 10min with TPBS, three 5min add chemical luminous substrate, the X-ray film developing.
Embodiment 1 and embodiment 2 come to the same thing, and experimental result has obtained good reappearance.
Claims (4)
1, a kind ofly changes 2 films simultaneously, the method of while labelled protein TRX and procaspase12, it is characterized in that: two films are labelled protein TRX and procaspase12 respectively, comprises extraction, protein quantification, gel electrophoresis, the Western blot of total protein of cell, the mark and the X-ray film developing step of antibody.
2, the method for claim 1 is characterized in that: extract total protein of cell, carry out gel electrophoresis behind protein quantification; Albumen with two overlapping being placed on the glue of onesize pvdf membrane, is carried out mark so that distinguish after gel electrophoresis, change the film condition and be: U=100V, I=350mA, T=70min; Antibody with two films difference mark TRX and procaspase12 carries out immunology detection, X-ray film developing testing result.
3, the method for claim 1 is characterized in that the dilution ratio of two kinds of antibody is respectively: TRX 1:5000, procaspase12 1:800.
4, the method for claim 1 is characterized in that extracting the PC12 total protein of cell, utilizes BCA method pair cell total protein quantitative; The total protein that extracts in the cell is carried out gel electrophoresis; With wet commentaries on classics method the albumen on the glue is transferred on two pvdf membranes simultaneously; One anti-mark, antigen-antibody is washed film with TPBS after hatching 70min; Two anti-marks are washed film with TPBS after hatching 70min with horseradish peroxidase two is anti-with antigen antibody complex; Add chemical luminous substrate and develop, obtain the band of two kinds of destination proteins; Check the repeated and stable of this method by traditional stripping method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100941697A CN101498734A (en) | 2009-03-09 | 2009-03-09 | Method for simultaneously turning two films and marking protein TRX and procaspase12 |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2009100941697A CN101498734A (en) | 2009-03-09 | 2009-03-09 | Method for simultaneously turning two films and marking protein TRX and procaspase12 |
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| CN101498734A true CN101498734A (en) | 2009-08-05 |
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| CNA2009100941697A Pending CN101498734A (en) | 2009-03-09 | 2009-03-09 | Method for simultaneously turning two films and marking protein TRX and procaspase12 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108225875A (en) * | 2018-01-15 | 2018-06-29 | 南通大学附属医院 | The preparation of nasal cavity washing liquid excretion body, identification method |
-
2009
- 2009-03-09 CN CNA2009100941697A patent/CN101498734A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| H TOWBIN ET AL.: "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications", 《PNAS》 * |
| 刘彬等: "半干式蛋白质电泳印迹的影响因素及条件优化", 《细胞生物学杂志》 * |
| 沈佐君等: "电泳技术在临床检验中的应用", 《中华检验医学杂志》 * |
| 董燕等: "电转移中蛋白质的透膜现象及其对蛋白质印迹结果的影响", 《生物化学与生物物理进展》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108225875A (en) * | 2018-01-15 | 2018-06-29 | 南通大学附属医院 | The preparation of nasal cavity washing liquid excretion body, identification method |
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Application publication date: 20090805 |