CN1262834C - Method for increasing resolution power and signal strength in protein gel electrolytic zone - Google Patents
Method for increasing resolution power and signal strength in protein gel electrolytic zone Download PDFInfo
- Publication number
- CN1262834C CN1262834C CN 200410066776 CN200410066776A CN1262834C CN 1262834 C CN1262834 C CN 1262834C CN 200410066776 CN200410066776 CN 200410066776 CN 200410066776 A CN200410066776 A CN 200410066776A CN 1262834 C CN1262834 C CN 1262834C
- Authority
- CN
- China
- Prior art keywords
- protein
- sample
- band
- homogenate
- colour developing
- 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.)
- Expired - Fee Related
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to the technical field of protein detection, particular to a method for increasing resolution power and signal strength in a protein gel electrolytic zone. The method comprises the steps: the preparation of protein samples, the separation of polypropylene gel electrophoresis and color display of a protein zone. The method is characterized in that electrophoretic separation are carried out to the detecting protein samples after the detecting protein samples are processed by acetone, and thereby, the color displaying signal strength of the protein is obviously strengthened. In addition, the method is favorable for the discovery of the protein with low abundance in the detecting samples, and thereby, the method provides convenience for further analytical judgment.
Description
Technical field
The present invention relates to the protein detection technology field, is a kind of antibody and display technique in conjunction with anti-respective egg white matter, can strengthen the observability of the protein band that gel electrophoresis separates and the method for signal intensity.
Background technology
The contained kinds of protein and the analysis and the comparison of protein molecule relative size in the various samples such as homogenate potpourri (liquid), lysis potpourri and serum for the preparation of the solid organ in animal or human source, polyacrylate hydrogel electrophoresis (as SDS-PAGE) is clinical at present and laboratory study technology commonly used.Through the polyacrylate hydrogel electrophoresis, can roughly each Separation of Proteins in the potpourri sample be come, again gel is aided with coomassie brilliant blue staining, can roughly differentiate the approximate location of the protein molecule that will separate.Yet the polyacrylate hydrogel electrophoresis is only separated the protein in the potpourri sample (as entity organ or serum), but can not change the relative content of a certain destination protein.If the abundance of a certain destination protein matter is very low, the signal of district's band of institute's isolated protein colour developing is very weak, just gives to detect with analysis and brings certain difficulty.The problem that this runs into through regular meeting with conventional sample processing method the time.
Summary of the invention
The invention provides a kind of method that strengthens gel electrophoresis of protein regional band resolution rate or strengthen transferring protein trace signal intensity.The present invention is the improvement to existing detection method.Crucial improvements are the detection sample liquid of preparation is carried out acetone treatment earlier, and then carry out electrophoretic separation and colour developing, the color signal that separates the protein band that obtains so obviously strengthens (no matter with coomassie brilliant blue staining or be transferred to nitrocellulose filter be aided with the chemiluminescence colour developing again), low abundance proteins might be found, and this just provides convenience for further analysis and judgement.The inventive method comprises the preparation of testing protein sample, the separation of polyacrylate hydrogel electrophoresis and the colour developing of protein band, it is characterized in that said testing protein sample is meant animal tissue's homogenate or cell culture fluid, bacterial lysate, animal body fluid (blood plasma, serum) the protein sample of crossing through acetone treatment, treatment conditions are: with homogenate or body fluid or nutrient solution or lysate centrifuging and taking supernatant, with supernatant and acetone with 1: 5 (v/v) mixing, put-20 ℃ more than 2 hours or spend the night, the centrifuging and taking precipitation, with resolution of precipitate in sample buffer [60mM Tris.Cl pH6.8,25% glycerine, 2% sodium dodecylsulphonate (SDS), 14.4mM 2 mercapto ethanol, 0.1% bromophenol blue], boiled 5 minutes, the centrifuging and taking supernatant is protein sample liquid; What said polyacrylate hydrogel electrophoresis was used is SDS-PAGE gel separation system, the about 80 μ g protein of every hole point sample amount; Said protein band colour developing is meant the protein band Western blotting and the chemiluminescence colour developing of coomassie brilliant blue staining or transfer.
Homogenate sample with the solid organ preparation is an example, and the concrete testing process of the present invention is as follows:
1. tissue homogenate preparation: routinely organ-tissue such as mouse spleen, lungs are shredded, fragment is put into homogenizer, add 4 ℃ and organize lysate to carry out homogenized.Organize lysate to be: 50mM Tris.Cl (pH8.0), 150mM NaCl, 0.02%NaN
3, 0.1%SDS, 1%NP-40,1 μ g/ml aprotinin (aprotinin), 100 μ g/ml PMSF, 1%2-mercaptoethanol.
2. the preliminary extraction and the acetone treatment of tissue protein:, draw supernatant with above-mentioned homogenate centrifugal (8000rpm, 4 ℃, centrifugal 5 minutes).With supernatant and acetone according to 1: 5 (v/v) mixing.This potpourri is-20 ℃ of placements at least 2 hours, with 10000rpm centrifugal 5 minutes, abandon supernatant, sediment.
3. the gel electrophoresis of protein separates: sediment is dissolved in sample buffer [60mM Tris.Cl pH6.8,25% glycerine, 2% sodium dodecylsulphonate (SDS), 14.4mM 2 mercapto ethanol, 0.1% bromophenol blue], boiled 5 minutes.The sample high speed centrifugation that will boil is got supernatant, measures the protein concentration in the supernatant, carries out protein electrophoresis with the SDS-PAGE gel systems and separates, and the point sample amount is that every hole contains 80 μ g protein approximately.And the above-mentioned tissue homogenate sample of crossing without acetone treatment carried out electrophoretic separation simultaneously on another clotting offset plate, with as experiment contrast.
4. coomassie brilliant blue staining: with Coomassie brilliant blue (Coomassie brilliant blue R-250) dyeing 30 minutes, dyeing liquor contained Coomassie brilliant blue 1 ‰, (w/v) with above-mentioned gel; 45% methyl alcohol, (v/v); 10% glacial acetic acid (v/v), decolours in destainer again, destainer contain 10% methyl alcohol, 10% glacial acetic acid, (v/v), till background is clear.
5. electrotransfer and Western blotting:
The protein that gel electrophoresis separates is transferred to (referring to the molecular cloning experiment guide) on the cellulose nitrate film, 350mA, 50min according to conventional electrotransfer method, shift formula of liquid: the 39mM glycocoll, 48mM Tris alkali, 0.037%SDS (electrophoresis level), 20% methyl alcohol.Put into confining liquid sealing 1.5h again, confining liquid is TBS-T (pH7.4) damping fluid that contains 5% skimmed milk power.On nitrocellulose filter, add the anti-mouse Toll of rabbit sample acceptor 2 (TLR-2) more routinely, caspase-3 and TNF (tumor necrosis factor) receptor associated factor (TRAF-6) antibody, the dilutability of every kind of antibody is 1: 800, put 4 ℃ 2 hours, with TBS-T (pH7.4) damping fluid with a unnecessary anti-flush away.The IgG (dilutability 1: 50) that adds goat-anti rabbit horseradish peroxidase (HRP) mark again, put 4 ℃ 2 hours, with TBS-T (pH7.4) damping fluid with unnecessary labelled antibody flush away.
6. the enhanced chemiluminescence Faxian shows the protein band of transfer: above-mentioned nitrocellulose filter is added chemiluminescence reaction liquid in the darkroom, cover the X-mating plate again on nitrocellulose filter, expose routinely, development, photographic fixing.After the X-mating plate dries, the protein band that shows is write down the result with scanner, row is analyzed again.
For humanized's sample, solid organ such as the spleen that retains as operation, kidney, liver, lung, the check and analysis method is the same.
Processing for the supernatant sample of blood and cellular incubation: can earlier serum, blood plasma or cell culture supernatant and acetone be mixed according to volume at 1: 5, all the other operations are the same.Protein for engineering bacteria gene expression is also handled with reference to said method.
Embodiment:
Now in conjunction with the embodiments the present invention is described in detail.
Reagent and material
(1) tissue lysate: 50mM Tris.Cl (pH8.0), 150mM-NaCl, 0.02%NaN
3, 0.1%SDS, 1%NP-40,1 μ g/ml aprotinin (aprotinin), 100 μ g/ml PMSF, 1%2-mercaptoethanol
(2) acetone treatment liquid: pure acetone, (the chemical reagent supply station, Shanghai) analyzed commonly used, laboratory
(3) chemical luminescence reagent kit: commercial order, Pufei Biotechnology Co. (China, Shanghai); X-ray film: Fuji's medical X-ray film (super-RX, Ref.03G050)
(4) the anti-mouse Toll of rabbit sample acceptor 2 (TLR-2), caspase-3 and TNF (tumor necrosis factor) receptor associated factor (TRAF-6) antibody: commercial order, Santa Cruz biotechnology (USA)
(5) goat anti-rabbit igg-HRP label and anti-mouse IgG-alkaline phosphatase enzyme conjugate are bought (China, Shanghai) from magnificent biotech company.
Embodiment 1. detects Caspase-3 in the mouse spleen homogenate sample.
1. prepare spleen homogenate sample.
Get mouse spleen, in the homogenizer that frozen water surrounds, carry out homogenized.The centrifuging and taking supernatant.The supernatant sample is divided into two parts, and a part is handled with conventional method, and another part is handled with the inventive method.
2. sample process
(1) conventional method is handled: with above-mentioned spleen homogenate supernatant, carry out determination of protein concentration (the Bradford method detects, down together), then certain density protein is mixed with the electrophoresis sample-loading buffer, and stand-by.
(2) the inventive method is handled: with spleen homogenate supernatant, press 1: 5 (v/v) mixing with acetone, put-20 ℃ again and spend the night.Centrifugal (centrifugal speed is 10000rpm) abandons supernatant, adds PBS (pH7.4) sediment is dissolved into the protein suspension.Measure the concentration of this suspension protein, then certain density protein is mixed with the electrophoresis sample-loading buffer, stand-by.
3. electrophoresis
The spleen homogenate sample liquid that two kinds of distinct methods of above-mentioned usefulness are handled application of sample respectively carries out electrophoretic separation in two blocks of polyacrylate hydrogel plates arranged side by side (direct current 100V, 1.5h), the application of sample amount is the protein of the about 80 μ g in every hole.
4. color development treatment:
Two kinds of disposal routes are arranged, and a kind of is with the gel direct staining, and another kind of is that gel moves on to colour developing again on the nitrocellulose filter by the electrotransfer method with protein transduction.
(1) direct staining of polyacrylate hydrogel:
Routinely above-mentioned gel is put into coomassie brilliant blue staining liquid dyeing 30 minutes, with the destainer decolouring, made background have contrast preferably again.
(2) electrophoresis band is transferred to nitrocellulose filter by polyacrylate hydrogel, carries out Western blotting again and the chemiluminescence of trace thing colour developing.
Method is: the protein band electricity consumption transfer method in the gel is transferred to cellulose nitrate film, again according to the Western blotting program, with an anti-(anti-mouse Caspase-3 of rabbit, antibody dilution is 1: 800) and nitrocellulose filter (hereinafter to be referred as transfer membrane) put into the micro plastic bag and carry out immuning hybridization reaction, 4 ℃, 2 hours.With TBS-T (pH7.4) washing transfer membrane, with the anti-mouse caspase-3IgG of unnecessary rabbit flush away.The goat anti-rabbit igg (dilutability is 1: 50) that adds the horseradish peroxidase mark then, carry out (4 ℃ of recognition reactions with transfer membrane, 2 hours), use TBS-T (pH7.4) washing 4 times again, each 3 minutes, behind the thorough flush away of goat anti-rabbit igg with unnecessary horseradish peroxidase mark, on transfer membrane, add the chemical illuminating reagent colour developing.Usually, add chemical illuminating reagent 0.125ml on every square centimeter the transfer membrane, make it to be layered on the transfer membrane equably.Transfer membrane is wrapped up with the food fresh keeping film in the darkroom rapidly, in protein face one side of transfer membrane, X-light sensation ray film carries out photoresponse in the covering.After finishing sensitization, sensitive film is developed and photographic fixing in the darkroom rapidly, manifest transferring protein district band.
TLR-2 in the detection mouse lung homogenate sample in Caspase-3 and spleen, the lung or the method for TRAF-6 are with embodiment 1.
The colour developing collection of illustrative plates of two kinds of disposal routes is seen Fig. 1, and the contrast of protein band colour developing density sees Table 1, table 2.
Table 1. the inventive method and conventional method are handled lung homogenate protein electrophorese district band density contrast table
| Zone of protein | The inventive method | Conventional method | Multiple |
| TLR-2 Caspase-3 TRAF-6 | 62.30 61.00 66.20 | 36.10 37.60 26.90 | 1.73 1.60 2.46 |
Table 2. the inventive method and conventional method are handled spleen homogenate protein electrophorese district band density contrast table
| Zone of protein | The inventive method | Conventional method | Multiple |
| TLR-2 Caspase-3 TRAF-6 | 82.30 64.50 59.00 | 15.30 35.00 38.30 | 5.37 1.84 1.54 |
Among Fig. 1, gel A, B, C use caspase-3, Toll sample acceptor 2 (TLR-2) and caspase-3, TLR-2, the TRAF-6 protein band of TNF (tumor necrosis factor) receptor associated factor (TRAF-6) antibody test transfer on nitrocellulose filter respectively.Swimming lane the 1, the 3rd, the sample that adopts the inventive method to handle, swimming lane the 2, the 4th, the sample that adopts conventional method to handle.Wherein swimming lane the 1, the 2nd, lungs homogenate sample, swimming lane the 3, the 4th, spleen homogenate sample.As seen from Figure 1, the inventive method is compared with the sample that conventional method is handled, and the abundance of its protein electrophorese district band obviously strengthens.After electrotransfer, Western blotting and enhanced chemiluminescence colour developing, the protein electrophorese district band of the sample that the protein electrophorese district band of the sample that the inventive method is handled is obviously handled than conventional method is dark.
Table 1 is that the inventive method and conventional method are handled lung homogenate protein electrophorese district band density contrast table.By table 1 as seen, in the albumen sample that lung homogenate is handled, the TLR-2 of separation strengthens 1.73 times than the signal of conventional sample process method; The caspase-3 signal strengthens 1.60 times; And the signal of the zone of protein of TRAF-6 strengthens 2.46 times.
Table 2 is that the inventive method and conventional method are handled spleen homogenate protein electrophorese district band density contrast table.By table 2 as seen, after the sample of spleen was handled through this method, the TLR-2 of separation strengthened 5.37 times than the signal of conventional sample process method; The caspase-3 signal strengthens 1.84 times; And the signal of the zone of protein of TRAF-6 strengthens 1.54 times.
This shows that method of the present invention can obviously strengthen protein band color signal intensity.
Claims (1)
1. method that can strengthen gel electrophoresis of protein regional band resolution rate and signal intensity, comprise the preparation of testing protein sample, the separation of polyacrylate hydrogel electrophoresis and the colour developing of protein band, it is characterized in that said testing protein sample is meant animal tissue's homogenate or cell culture fluid, bacterial lysate, the protein sample that animal body fluid is crossed through acetone treatment, treatment conditions are: with homogenate or body fluid or nutrient solution or lysate centrifuging and taking supernatant, with supernatant and acetone with 1: 5 volume ratio mixing, put-20 ℃ more than 2 hours or spend the night, the centrifuging and taking precipitation, is 60mM Tris.Cl pH6.8 with resolution of precipitate in composition, 25% glycerine, 2% sodium dodecylsulphonate, 14.4mM 2 mercapto ethanol, the sample buffer of 0.1% bromophenol blue was boiled 5 minutes, and the centrifuging and taking supernatant is protein sample liquid; What said polyacrylate hydrogel electrophoresis was used is SDS-PAGE gel separation system, every hole point sample amount 80 μ g protein; Said protein band colour developing is meant the protein band Western blotting and the chemiluminescence colour developing of coomassie brilliant blue staining or transfer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410066776 CN1262834C (en) | 2004-09-29 | 2004-09-29 | Method for increasing resolution power and signal strength in protein gel electrolytic zone |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410066776 CN1262834C (en) | 2004-09-29 | 2004-09-29 | Method for increasing resolution power and signal strength in protein gel electrolytic zone |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1588032A CN1588032A (en) | 2005-03-02 |
| CN1262834C true CN1262834C (en) | 2006-07-05 |
Family
ID=34604091
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410066776 Expired - Fee Related CN1262834C (en) | 2004-09-29 | 2004-09-29 | Method for increasing resolution power and signal strength in protein gel electrolytic zone |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1262834C (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100573093C (en) * | 2006-08-02 | 2009-12-23 | 中国科学院植物研究所 | A kind of coomassie brilliant blue staining method and special gel immobile liquid and coloring agent |
| CN101750413B (en) * | 2009-11-09 | 2011-05-04 | 河南工业大学 | Rapid detection method for protein content of foods |
| CN104004382B (en) * | 2014-05-20 | 2016-01-20 | 北京五康新兴科技有限公司 | A kind of coomassie brilliant blue staining liquid and dyeing process |
| CN107271657B (en) * | 2016-04-08 | 2018-06-05 | 北京爱普拜生物技术有限公司 | A kind of protein immunoblotting signal enhancing agent |
| CN107228894B (en) * | 2017-01-24 | 2019-03-19 | 浙江海隆生物科技有限公司 | Method for detecting protein content and purity in oil emulsion protein subunit vaccine and application thereof |
| CN110320086B (en) * | 2019-07-09 | 2020-04-14 | 上海宝藤生物医药科技股份有限公司 | Lipoprotein electrophoresis staining solution and preparation method and application thereof |
-
2004
- 2004-09-29 CN CN 200410066776 patent/CN1262834C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1588032A (en) | 2005-03-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kremser et al. | Capillary electrophoresis of biological particles: viruses, bacteria, and eukaryotic cells | |
| Ahmed et al. | Strategies for revealing lower abundance proteins in two-dimensional protein maps | |
| Kostal et al. | Recent advances in the analysis of biological particles by capillary electrophoresis | |
| CN102421799B (en) | Novel monoclonal antibody for analyzing high-molecular weight adiponectin and utilization of same | |
| US20230393042A1 (en) | Pretreatment method, preservation method, automatic treatment system and detection method for urine sample | |
| CN112029822A (en) | Small and dense low-density lipoprotein cholesterol enzyme method detection kit | |
| CN1262834C (en) | Method for increasing resolution power and signal strength in protein gel electrolytic zone | |
| Karlsson et al. | Quantitation of proteoglycans in biological fluids using Alcian blue | |
| Karlsson et al. | Binding and detection of glycosaminoglycans immobilized on membranes treated with cationic detergents | |
| CN111239271A (en) | Method for quantifying trace biological sample proteome by utilizing isotope labeling technology | |
| CN106496328A (en) | The preparation method of hyaluronic acid binding protein polyclonal antibody | |
| CN111100839B (en) | EGFR/Vimentin/folic acid immunoliposome magnetic sphere, preparation method and kit | |
| CN113960313B (en) | Exosome ALK fusion protein magnetic immunochemiluminescence detection kit | |
| Wilson et al. | Preparative isoelectric focusing of immunologically reactive components of Aspergillus fumigatus mycelium | |
| Govorun et al. | Proteomics and peptidomics in fundamental and applied medical studies | |
| CN102445487B (en) | Electrophoretic analysis method for modified protein isolation and identification | |
| US20240192164A1 (en) | Method for enhancing signals associated with electrophoretically separated analytes using post-electrophoresis treatment | |
| CN104478999A (en) | Method of extracting bovine mycoplasma outer membrane protein suitable for proteomics | |
| Kilár et al. | Novel quantitative electrophoretic analysis of endotoxins on microchips | |
| CN116875604B (en) | DNA aptamer for detecting PD-L1 positive cells and application thereof | |
| JP2003502665A (en) | Capillary electrophoresis for affinity ligand screening using competing detectable ligands | |
| CN112034176B (en) | Use of CNTN1 as molecular marker for long-term low-dose ionizing radiation exposure diagnosis | |
| CN108007997A (en) | A kind of shiitake mushroom hypha total protein extraction and separation method for dielectrophoresis | |
| CN110437341B (en) | Detection protein with red fluorescence activity and application thereof | |
| Kilgore et al. | The determination of specific radioactivity of proteins eluted intact from polyacrylamide gels, utilizing a fluorescamine assay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |