CN114486456B - Protein staining kit for polyacrylamide gel electrophoresis and staining method thereof - Google Patents
Protein staining kit for polyacrylamide gel electrophoresis and staining method thereof Download PDFInfo
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 43
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 43
- 238000010186 staining Methods 0.000 title claims abstract description 15
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 title claims abstract description 10
- 238000007447 staining method Methods 0.000 title abstract description 8
- 239000000243 solution Substances 0.000 claims abstract description 56
- 238000000034 method Methods 0.000 claims abstract description 32
- 229920002401 polyacrylamide Polymers 0.000 claims abstract description 30
- 238000001962 electrophoresis Methods 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 12
- 238000005406 washing Methods 0.000 claims abstract description 12
- 125000003396 thiol group Chemical group [H]S* 0.000 claims abstract description 10
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 8
- 239000007864 aqueous solution Substances 0.000 claims abstract description 3
- 239000002904 solvent Substances 0.000 claims abstract description 3
- 235000018102 proteins Nutrition 0.000 claims description 35
- 238000004043 dyeing Methods 0.000 claims description 20
- 238000010438 heat treatment Methods 0.000 claims description 11
- 229910052751 metal Inorganic materials 0.000 claims description 8
- 239000002184 metal Substances 0.000 claims description 8
- 108010024636 Glutathione Proteins 0.000 claims description 5
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 5
- -1 halogen salt Chemical class 0.000 claims description 5
- AKIZPWSPNKVOMT-UHFFFAOYSA-N 1-sulfanylhexan-1-ol Chemical compound CCCCCC(O)S AKIZPWSPNKVOMT-UHFFFAOYSA-N 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 4
- 235000018417 cysteine Nutrition 0.000 claims description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 3
- 229910002651 NO3 Inorganic materials 0.000 claims description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 3
- 229910052736 halogen Inorganic materials 0.000 claims description 3
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 claims description 3
- HKNNAYPWWDWHFR-UHFFFAOYSA-N 1-sulfanylbutan-1-ol Chemical compound CCCC(O)S HKNNAYPWWDWHFR-UHFFFAOYSA-N 0.000 claims description 2
- XBCDECDLTNTPSZ-UHFFFAOYSA-N 1-sulfanylheptan-1-ol Chemical compound CCCCCCC(O)S XBCDECDLTNTPSZ-UHFFFAOYSA-N 0.000 claims description 2
- ILWVAHWMSULXIG-UHFFFAOYSA-N 1-sulfanyloctan-1-ol Chemical compound CCCCCCCC(O)S ILWVAHWMSULXIG-UHFFFAOYSA-N 0.000 claims description 2
- PDXOPTFTOFXXSU-UHFFFAOYSA-N 1-sulfanylpentan-1-ol Chemical compound CCCCC(O)S PDXOPTFTOFXXSU-UHFFFAOYSA-N 0.000 claims description 2
- AEUVIXACNOXTBX-UHFFFAOYSA-N 1-sulfanylpropan-1-ol Chemical compound CCC(O)S AEUVIXACNOXTBX-UHFFFAOYSA-N 0.000 claims description 2
- OMEBWCFMNYDCFP-UHFFFAOYSA-N 1-sulfanylundecan-1-ol Chemical compound CCCCCCCCCCC(O)S OMEBWCFMNYDCFP-UHFFFAOYSA-N 0.000 claims description 2
- CFPHMAVQAJGVPV-UHFFFAOYSA-N 2-sulfanylbutanoic acid Chemical compound CCC(S)C(O)=O CFPHMAVQAJGVPV-UHFFFAOYSA-N 0.000 claims description 2
- BZGHIQTWNAKSCX-UHFFFAOYSA-N 2-sulfanylheptanoic acid Chemical compound CCCCCC(S)C(O)=O BZGHIQTWNAKSCX-UHFFFAOYSA-N 0.000 claims description 2
- JSGPBRQYMLFVJQ-UHFFFAOYSA-N 2-sulfanylhexanoic acid Chemical compound CCCCC(S)C(O)=O JSGPBRQYMLFVJQ-UHFFFAOYSA-N 0.000 claims description 2
- WXVYGDSYOULXDD-UHFFFAOYSA-N 2-sulfanyloctanoic acid Chemical compound CCCCCCC(S)C(O)=O WXVYGDSYOULXDD-UHFFFAOYSA-N 0.000 claims description 2
- IMGGANUNCHXAQF-UHFFFAOYSA-N 2-sulfanylpentanoic acid Chemical compound CCCC(S)C(O)=O IMGGANUNCHXAQF-UHFFFAOYSA-N 0.000 claims description 2
- DYAOREPNYXXCOA-UHFFFAOYSA-N 2-sulfanylundecanoic acid Chemical compound CCCCCCCCCC(S)C(O)=O DYAOREPNYXXCOA-UHFFFAOYSA-N 0.000 claims description 2
- IYGAMTQMILRCCI-UHFFFAOYSA-N 3-aminopropane-1-thiol Chemical compound NCCCS IYGAMTQMILRCCI-UHFFFAOYSA-N 0.000 claims description 2
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 claims description 2
- RIRRYXTXJAZPMP-UHFFFAOYSA-N 4-aminobutane-1-thiol Chemical compound NCCCCS RIRRYXTXJAZPMP-UHFFFAOYSA-N 0.000 claims description 2
- DXEUCMHRAHWVEF-UHFFFAOYSA-N 5-aminopentane-1-thiol Chemical compound NCCCCCS DXEUCMHRAHWVEF-UHFFFAOYSA-N 0.000 claims description 2
- WYYXDSQOPIGZPU-UHFFFAOYSA-N 6-aminohexane-1-thiol Chemical compound NCCCCCCS WYYXDSQOPIGZPU-UHFFFAOYSA-N 0.000 claims description 2
- VFZBISABHRHOEL-UHFFFAOYSA-N 7-aminoheptane-1-thiol Chemical compound NCCCCCCCS VFZBISABHRHOEL-UHFFFAOYSA-N 0.000 claims description 2
- BWUPKMHSAHUZGN-UHFFFAOYSA-N 8-aminooctane-1-thiol Chemical compound NCCCCCCCCS BWUPKMHSAHUZGN-UHFFFAOYSA-N 0.000 claims description 2
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 claims description 2
- 150000004662 dithiols Chemical class 0.000 claims description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 2
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims description 2
- 239000010931 gold Substances 0.000 claims description 2
- 229910052737 gold Inorganic materials 0.000 claims description 2
- 229960003151 mercaptamine Drugs 0.000 claims description 2
- RSPCKAHMRANGJZ-UHFFFAOYSA-N thiohydroxylamine Chemical compound SN RSPCKAHMRANGJZ-UHFFFAOYSA-N 0.000 claims description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 2
- 150000001412 amines Chemical class 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 10
- 238000004458 analytical method Methods 0.000 abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 4
- 238000002331 protein detection Methods 0.000 abstract description 2
- 239000002699 waste material Substances 0.000 abstract description 2
- 239000000499 gel Substances 0.000 description 60
- 230000000052 comparative effect Effects 0.000 description 11
- 239000012192 staining solution Substances 0.000 description 10
- 238000011534 incubation Methods 0.000 description 8
- 238000002791 soaking Methods 0.000 description 8
- 239000012474 protein marker Substances 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 239000008367 deionised water Substances 0.000 description 6
- 229910021641 deionized water Inorganic materials 0.000 description 6
- 238000011010 flushing procedure Methods 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000010979 ruby Substances 0.000 description 5
- 229910001750 ruby Inorganic materials 0.000 description 5
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 208000002109 Argyria Diseases 0.000 description 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229910052709 silver Inorganic materials 0.000 description 2
- 239000004332 silver Substances 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- PJHWBEFCLFGUQX-UHFFFAOYSA-N 1-aminoundecane-1-thiol Chemical compound CCCCCCCCCCC(N)S PJHWBEFCLFGUQX-UHFFFAOYSA-N 0.000 description 1
- DIXRLQJYISYSEL-UHFFFAOYSA-N 11-aminoundecane-1-thiol Chemical compound NCCCCCCCCCCCS DIXRLQJYISYSEL-UHFFFAOYSA-N 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- OTCKNHQTLOBDDD-UHFFFAOYSA-K gold(3+);triacetate Chemical compound [Au+3].CC([O-])=O.CC([O-])=O.CC([O-])=O OTCKNHQTLOBDDD-UHFFFAOYSA-K 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000010815 organic waste Substances 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
Abstract
The invention belongs to the technical field of protein detection, and discloses a protein staining kit for polyacrylamide gel electrophoresis and a staining method thereof. The kit comprises a solution A and a solution B, wherein the solution A comprises metal ions, the solution B comprises an aqueous solution containing a reducing molecule, the reducing molecule contains sulfhydryl groups, and the reagent is not easy to volatilize and does not generate waste liquid, so that the kit is friendly to experimental operators and environment; the sensitivity to protein staining is high, and the comprehensive cost is low. The staining method of the kit comprises the following steps: after electrophoresis, placing the polyacrylamide gel in the solution A, incubating for 1-60 min, and washing the polyacrylamide gel with water; and (3) placing the polyacrylamide gel in the solution B, incubating for 1-60 min, and washing the polyacrylamide gel with water. The method is simple and convenient to operate, and the quick and pollution-free polyacrylamide gel electrophoresis protein staining analysis is realized by using water as the only solvent.
Description
Technical Field
The invention belongs to the technical field of protein detection, and particularly relates to a protein staining kit for polyacrylamide gel electrophoresis and a staining method thereof.
Background
Proteins are the material basis of life, are organic macromolecules, and are the main contributors to life activities. Proteins exert various functions in organisms, such as catalytic functions, transport functions, mechanical support and protection functions, regulatory functions, immune and defensive functions, etc., and are particularly important as a undertaker of various functions.
Investigation of the morphology and structure of proteins often requires extraction of the protein, followed by isolation of a single species of protein for investigation. Polyacrylamide gel electrophoresis is a commonly used protein separation method at present, can separate proteins and determine the relative molecular weight and the protein purity of the proteins, and can also be used for separating and purifying the proteins. After electrophoresis, the protein can be subjected to corresponding analysis and research by dyeing the gel, the dyeing methods comprise a plurality of types, such as coomassie brilliant blue dyeing method, sypro Ruby dyeing method, silver dyeing method and the like, the dyeing methods take a long time from the end of electrophoresis to the completion of imaging, the steps are complicated, formaldehyde-type volatile and toxic reagents such as methanol, acetic acid and formaldehyde are often used in the process, and the whole process needs repeated washing and decoloring to generate a large amount of organic waste liquid, so that the method is not friendly to experimental operators and environment. The sensitivity of these methods is related to the complexity of the operation and the time consumption, and the higher the sensitivity is, the more complex the operation is, the longer the time consumption is, and the satisfactory result cannot be obtained quickly. Therefore, it is important to provide a protein staining analysis method which can achieve high speed and high sensitivity.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a protein staining kit for polyacrylamide gel electrophoresis, which can quickly stain the protein in the gel with high sensitivity and no pollution.
The invention also provides a dyeing method of the dyeing kit.
According to one aspect of the present invention, there is provided a protein staining kit for polyacrylamide gel electrophoresis, comprising a solution a comprising metal ions and a solution B comprising an aqueous solution containing a reducing molecule having a thiol group.
According to a preferred embodiment of the invention, there is at least the following advantageous effect:
the reagent used by the dyeing kit is not easy to volatilize, does not generate waste liquid, and is friendly to experimental operators and environment; the sensitivity to protein staining is high, and the comprehensive cost is low.
In some embodiments of the invention, the thiol group may reduce the metal ion to a metal atom that nucleates to grow to form a fluorescent metal nanocluster when the solution a and the solution B are mixed.
In some embodiments of the present invention, the volume part of the liquid a is 10 to 30 parts, and the volume part of the liquid B is 10 to 30 parts.
In some embodiments of the invention, the concentration of the solution A is 0.5mM to 5mM.
In some preferred embodiments of the invention, the concentration of solution A is 2mM.
In some embodiments of the invention, the concentration of the solution B is 5 mM-30 mM.
In some preferred embodiments of the invention, the concentration of solution B is 20mM.
In some embodiments of the invention, the liquid a comprises at least one of a halogen salt, nitrate or acetate of a metal ion.
In some preferred embodiments of the present invention, the liquid a includes at least one of a halogen salt, nitrate or acetate of gold ion or silver ion.
In some more preferred embodiments of the invention, the solution a comprises chloroauric acid.
In some embodiments of the invention, the solution B comprises at least one of cysteine, reduced glutathione, dithiol, mercapto acid, mercapto alcohol, or mercapto amine.
In some preferred embodiments of the present invention, the solution B comprises at least one of cysteine, reduced glutathione, dithiothreitol, thioglycolic acid, mercaptoethanol, mercaptoethylamine, mercaptopropionic acid, mercaptopropanol, mercaptopropylamine, mercaptobutyric acid, mercaptobutanol, mercaptobutylamine, mercaptovaleric acid, mercaptopentanol, mercaptopentylamine, mercaptohexanoic acid, mercaptohexanol, mercaptohexylamine, mercaptoheptanoic acid, mercaptoheptanol, mercaptoheptylamine, mercaptooctanoic acid, mercaptooctanol, mercaptooctylamine, mercaptoundecanoic acid, mercaptoundecanol, or mercaptoundecylamine.
In some more preferred embodiments of the invention, the liquid B comprises mercaptohexanol.
According to a further aspect of the present invention, a method of staining the staining kit is presented, comprising the steps of:
s1: after electrophoresis, placing the polyacrylamide gel in the solution A, incubating for 1-60 min, and washing the polyacrylamide gel with water;
s2: and (3) placing the polyacrylamide gel in the step (S1) in the solution B, incubating for 1-60 min, and washing the polyacrylamide gel with water.
The dyeing method according to a preferred embodiment of the present invention has at least the following advantageous effects:
the dyeing method adopted by the invention has simple principle, namely, after the protein to be detected is taken as a template and the protein is combined with the metal ions in the solution A, the sulfhydryl in the solution B can reduce the metal ions into metal atoms, and the metal atoms nucleate and grow on the protein to form fluorescent metal nanoclusters, so that the detection sensitivity is improved; the operation flow is simple and convenient, only water is used as a unique solvent, a simple 'soaking-rinsing' two-step method is formed, heating is not needed, cleaning is convenient, and quick and pollution-free polyacrylamide gel electrophoresis protein staining analysis is realized.
In some embodiments of the invention, the electrophoresis of step S1 comprises adding a protein sample to a polyacrylamide gel, and activating a power source to separate proteins in the protein sample from the polyacrylamide gel according to their molecular weight.
In some embodiments of the invention, the incubation described in step S1 is performed using a shaking incubation method.
In some embodiments of the invention, the incubation described in step S1 is performed for a period of time ranging from 1min to 10min.
In some preferred embodiments of the invention, the incubation described in step S1 is performed for a specific period of 5 minutes.
In some embodiments of the invention, the incubation described in step S2 is performed using a shaking incubation method.
In some embodiments of the invention, the incubation described in step S2 is performed for a period of time ranging from 5min to 15min.
In some preferred embodiments of the invention, the incubation described in step S2 is performed for a specific time period of 10min.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1
The embodiment adopts the kit and the method to dye the protein standard substance, and the specific process is as follows:
(1) Placing polyacrylamide gel in an electrophoresis tank, adding 10 mu L of standard protein Marker (Thermo, 26616) into a sample hole of the polyacrylamide gel, starting a power supply, regulating current to 240mA, and carrying out electrophoresis for 30min;
(2) Preparing a staining solution: according to the volume parts, 15 parts of chloroauric acid with the concentration of 2mM is prepared into solution A, and 20 parts of mercaptohexanol with the concentration of 20mM is prepared into solution B for standby;
(3) After electrophoresis, taking out the gel, soaking the gel in the solution A, placing the gel on a constant temperature shaking table for shaking and incubating for 5min, removing the solution A, and flushing the gel with a small amount of deionized water;
(4) Soaking the gel in the solution B, placing on a constant temperature shaking table, shaking and incubating for 10min, removing the solution B, and flushing the gel with a small amount of deionized water.
Example 2
The embodiment adopts the kit and the method to dye the protein standard substance, and the specific process is as follows:
(1) Placing polyacrylamide gel in an electrophoresis tank, adding 10 mu L of standard protein Marker (Thermo, 26616) into a sample hole of the polyacrylamide gel, starting a power supply, regulating current to 240mA, and carrying out electrophoresis for 30min;
(2) Preparing a staining solution: according to the volume parts, 15 parts of silver nitrate with the concentration of 2mM is prepared into solution A, and 20 parts of mercaptoundecanamine with the concentration of 20mM is prepared into solution B for standby;
(3) After electrophoresis, taking out the gel, soaking the gel in the solution A, placing the gel on a constant temperature shaking table for shaking and incubating for 5min, removing the solution A, and flushing the gel with a small amount of deionized water;
(4) Soaking the gel in the solution B, placing on a constant temperature shaking table, shaking and incubating for 10min, removing the solution B, and flushing the poly gel with a small amount of deionized water.
Example 3
The embodiment adopts the kit and the method to dye the protein standard substance, and the specific process is as follows:
(1) Placing polyacrylamide gel in an electrophoresis tank, adding 10 mu L of standard protein Marker (Thermo, 26616) into a sample hole of the polyacrylamide gel, starting a power supply, regulating current to 240mA, and carrying out electrophoresis for 30min;
(2) Preparing a staining solution: according to the volume parts, 15 parts of gold acetate with the concentration of 2mM is prepared into solution A, and 20 parts of reduced glutathione with the concentration of 20mM is prepared into solution B for standby;
(3) After electrophoresis, taking out the gel, soaking the gel in the solution A, placing the gel on a constant temperature shaking table for shaking and incubating for 5min, removing the solution A, and flushing the gel with a small amount of deionized water;
(4) Soaking the gel in the solution B, placing on a constant temperature shaking table, shaking and incubating for 10min, removing the solution B, and flushing the gel with a small amount of deionized water.
Comparative example 1
The comparative example adopts a coomassie brilliant blue staining method to stain a protein standard substance, and comprises the following specific processes:
(1) Placing polyacrylamide gel in an electrophoresis tank, adding 10 mu L of standard protein Marker (Thermo, 26616) into a sample hole of the polyacrylamide gel, starting a power supply, regulating current to 240mA, and carrying out electrophoresis for 30min;
(2) Preparing a staining solution: commercial coomassie brilliant blue staining solution (bi yun, P0017B);
(3) After electrophoresis, taking out gel, soaking the gel in coomassie brilliant blue staining solution, heating to just boiling by a microwave oven, immediately stopping heating, and placing on a constant-temperature shaking table for shaking and incubating for 5min;
(4) Transferring the gel into decolorizing solution, heating in a microwave oven until the gel just boils, immediately stopping heating, and placing on a constant temperature shaking table for shaking and incubating for 5min;
(5) And (3) replacing the fresh decolorizing liquid, and repeating the step (4) until the blue background is substantially completely removed.
Comparative example 2
The comparative example adopts a Sypro Ruby staining method to stain a protein standard substance, and the specific process is as follows:
(1) Placing polyacrylamide gel in an electrophoresis tank, adding 10 mu L of standard protein Marker (Thermo, 26616) into a sample hole of the polyacrylamide gel, starting a power supply, regulating current to 240mA, and carrying out electrophoresis for 30min;
(2) Preparing a staining solution: commercially availableRuby protein gel staining solution (Thermo Fisher, S12001);
(3) After electrophoresis, taking out gel, soaking the gel in a fixing solution, heating by a microwave oven, oscillating and incubating for 15min, removing the fixing solution, replacing fresh fixing solution and repeatedly heating once;
(4) Placing the gel onHeating in a Ruby protein gel staining solution for 30s in a microwave oven, stirring for 30s to uniformly heat, continuously heating for 30s to 80-85 ℃, and placing on a constant temperature shaking table for shaking and incubating for 5min; heating for 30s again, and incubating for 23min under shaking;
(5) Transferring the gel into a washing liquid to wash for 30min; finally, the solution is washed twice with ultrapure water for 5min each time.
Comparative example 3
The comparative example adopts a silver staining method to stain a protein standard substance, and comprises the following specific processes:
(1) Placing polyacrylamide gel in an electrophoresis tank, adding 10 mu L of standard protein Marker (Thermo, 26616) into a sample hole of the polyacrylamide gel, starting a power supply, regulating current to 240mA, and carrying out electrophoresis for 30min;
(2) Preparing a staining solution: commercial Pierce silver staining kit (Thermo SCIENTIFIC, 24612);
(3) After electrophoresis, taking out the polyacrylamide gel, placing the polyacrylamide gel in ultrapure water, washing for 5min, and repeating for one time;
(4) Fixing the gel in the fixing solution for 2 times for 15min each time; washing with 10% ethanol for 2 times each for 5min, and then with ultrapure water for 2 times each for 5min;
(5) Transferring the gel into sensitization working solution for 1min, and then washing with water for 2 times for 1min each time; placing the gel in a dyeing working solution for dyeing for 30min, then washing the gel with ultrapure water for 2 times, each time for 20s, and then placing the gel in a developer working solution for 2-3 min until a strip appears; finally, 5% acetic acid is added for 10min, and the color development is stopped (the steps (3) to (5) are performed on a constant temperature shaking table).
Test examples
The protein bands in the gels stained in the examples and comparative examples were analyzed in this test example.
The specific method comprises the following steps: the polyacrylamide gels of examples 1, 2 and comparative examples 1, 2 and 3 were taken, placed in a gel imaging analyzer, observed, photographed, and analyzed for protein staining sensitivity in the gel with additional software, and the results are shown in table 1.
TABLE 1
| Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 | |
| Sensitivity of | 10pg | 200pg | 20pg | 5ng | 500pg | 250pg |
As can be seen from Table 1, the kit and the corresponding method of the scheme are used for dyeing the standard protein Marker in the polyacrylamide gel, and can detect 10pg of protein bands, which are far lower than protein bands detected by using a Coomassie brilliant blue dyeing method, a Sypro Ruby dyeing method and a silver dyeing method, so that the kit and the dyeing method of the scheme are simple to operate and high in protein dyeing sensitivity.
While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (4)
1. A protein staining kit for polyacrylamide gel electrophoresis, comprising a solution a and a solution B, wherein the solution a comprises metal ions, and the solution B comprises an aqueous solution containing a reducing molecule, and the reducing molecule has a sulfhydryl group; the dyeing method of the kit only uses water as a unique solvent to form a 'soaking-rinsing' two-step method without heating, and comprises the following steps of:
s1: after electrophoresis, placing the polyacrylamide gel in the solution A, incubating for 1-10 min, and washing the polyacrylamide gel with water;
s2: placing the polyacrylamide gel in the step S1 into the solution B, incubating for 5-15 min, and washing the polyacrylamide gel with water;
when the solution A and the solution B are mixed, the sulfhydryl can reduce the metal ions into metal atoms, and the metal atoms nucleate and grow to form fluorescent metal nanoclusters;
the solution A comprises at least one of halogen salt, nitrate or acetate of gold ions.
2. The kit according to claim 1, wherein the volume part of the liquid a is 10 to 30 parts and the volume part of the liquid B is 10 to 30 parts.
3. The kit of claim 1, wherein the solution B comprises at least one of cysteine, reduced glutathione, dithiol, mercapto acid, mercapto alcohol, or mercapto amine.
4. The kit of claim 1, wherein the solution B comprises at least one of cysteine, reduced glutathione, dithiothreitol, thioglycollic acid, mercaptoethanol, mercaptoethylamine, mercaptopropionic acid, mercaptopropanol, mercaptopropylamine, mercaptobutyric acid, mercaptobutanol, mercaptobutylamine, mercaptovaleric acid, mercaptopentanol, mercaptopentylamine, mercaptohexanoic acid, mercaptohexanol, mercaptohexylamine, mercaptoheptanoic acid, mercaptoheptanol, mercaptoheptylamine, mercaptooctanoic acid, mercaptooctanol, mercaptooctylamine, mercaptoundecanoic acid, mercaptoundecanol, or mercaptoundecanoic amine.
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