CN1904578A - Kaomas brilliant blue dyeing method and its special gel fixation liquid and dyeing agent - Google Patents
Kaomas brilliant blue dyeing method and its special gel fixation liquid and dyeing agent Download PDFInfo
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- CN1904578A CN1904578A CN 200610089093 CN200610089093A CN1904578A CN 1904578 A CN1904578 A CN 1904578A CN 200610089093 CN200610089093 CN 200610089093 CN 200610089093 A CN200610089093 A CN 200610089093A CN 1904578 A CN1904578 A CN 1904578A
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Abstract
The invention discloses a Coomassie brilliant blue dyeing method and its special gel stationary liquid and coloring agent. It includes the following steps: putting SDS-PAGE glue into the gel stationary liquid for 0.5-2.0h at normal temperature; adding the glue in acetate solution with 0.5-3.0% volume percent concentration to extend for 1.0-3.0h at normal temperature; adding it to the Coomassie brilliant blue coloring agent to dye for 8-24h at normal temperature; add it to decolorizing solution I to decolorize for 1-3h; then adding to decolorizing solution II to decolorize to clear. The invention can detect SDS-PAGE glue protein at nanogram.
Description
Technical field
The present invention relates to a kind of coomassie brilliant blue staining method and special gel immobile liquid and coloring agent.
Background technology
Though in proteomics research, there are a variety of protein staining methods available at present,, still coomassie brilliant blue staining method (examining the method for dying), argentation and the fluorescence colour that are most widely used.Because it is highly sensitive, running program is easy relatively, the fluorescent dye of SYPRO family becomes the most popular dyestuff (T.H.Steinberg at present, L.J.Jones, R.P.Haugland, V.L.Singer, SYPRO Orange and SYPRO Red ProteinGel Stains:One-Step Fluorescent Staining of Denaturing Gels for Detectionof Nanogram Levels of Protein, Anal.Biochem.239 (1996) 223-227; M.F.Lopez, K.Berggren, E.Chernokalskaya, A.Lazarev, M.Robinson, W.F.Patton, Acomparison of silver stain and SYPRO Ruby Protein Gel Stain with respect toprotein detection in two-dimensional gels and identification by peptide massprofiling.Electrophoresis 21 (2000) 3673-3683.).Recently, the staining technique of a kind of DIGE by name also is used to detect the differentially expressed protein (W.F.Patton on the 2-DE glue, A thousand points oflight:the application of fluorescence detection technologies totwo-dimensional gel electrophoresis and proteomics, Electrophoresis 21 (2000) 1123-1144.).Yet, blemish in an otherwise perfect thing be these fluorescent dyes cost an arm and a leg, and fluorescence usually a few minutes with regard to cancellation.In addition, the fluorescent dye The Application of Technology be unable to do without special-purpose instrument and equipment and special protein quantification software.Argentation is a kind of the sensitiveest method of protein detection except that radioactive label of generally acknowledging at present, can be at the protein that detects on the nanogram level level on the SDS-PAGE glue, but, because complex steps, it is high that operation is wanted, careless slightly, will cause very dark background, influence result's sharpness and resolution.Most serious of all, a lot of silver dye glue decolouring difficulty, aldehyde materials such as used glutaraldehyde can carry out irreversible modification to protein, the serious mass spectrum evaluation work of disturbing the later stage, therefore, in proteomics research, do not recommend usually to use silver to dye (G.Candiano, M.Bruschi, L.Musante, L.Santucci, G.M.Ghiggeri, B.Carnemolla, P.Orecchia, L.Zardi, P.G.Righetti, Blue silver:A very sensitive colloidal CoomassieG-250 staining for proteome analysis, Electrophoresis 25 (2004) 1327-1333.).
In view of the deficiency of above-mentioned argentation and fluorescence colour, many researchists are more prone to adopt comparatively classical coomassie brilliant blue staining method when carrying out proteomics research.Examining the method for dying and why be subjected to everybody favor, mainly is because it has good repeatability, extremely low dyeing background, higher sensitivity (about 0.5 μ g/mm
2) with and good mass spectrum compatibility.Particularly pass through after people's such as Neuhoff the initiative work, the sensitivity of examining the method for dying is significantly improved, can detect the band (V.Neuhoff that total protein is about 30 nanograms, A.Norbert, D.Taube, W.Ehrhardt, Improved staining of proteins inpolyacrylamide gels including isoelectric focusing gels with clear backgroundat nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250, Electrophoresis 9 (1988) 255-262.).Subsequently, on people's such as Neuhoff research basis, Candiano and co-worker thereof have invented the dyeing method of examining of a kind of Blue of being called as Silver, this method can detect the band that only contains 1 nanogram albumen, sensitivity is near argentation, it is said be reported at present examine the highest a kind of of dyeing method medium sensitivity, the long time but this method need be decoloured in water makes the glue suction expand, become and be highly brittle, (G.Candiano, M.Bruschi, L.Musante very easily break, L.Santucci, G.M.Ghiggeri, B.Carnemolla, P.Orecchia, L.Zardi, P.G.Righetti, Blue silver:A very sensitivecolloidal Coomassie G-250 staining for proteome analysis, Electrophoresis 25 (2004) 1327-1333.).
Summary of the invention
The purpose of this invention is to provide the coomassie brilliant blue staining method in a kind of proteomics research field and migrate gel stationary liquid and coloring agent.
Gel stationary liquid provided by the present invention is that to contain concentration expressed in percentage by volume be 8-12% acetic acid, 30-50% ethanol and 5-15% methanol in water.
The content of the acetic acid in the above-mentioned gel stationary liquid is preferably 10%, and the content of ethanol is preferably 40%, and the content of methyl alcohol is preferably 10%.
Coomassie brilliant blue staining agent provided by the present invention is that to contain concentration expressed in percentage by volume be 4-6% acetic acid, and 35-55% ethanol and mass volume concentrations are the aqueous solution of 0.1-0.15% Coomassie brilliant blue R-250.
The content of the acetic acid in the above-mentioned coomassie brilliant blue staining agent is preferably 5%, and the content of ethanol is preferably 45%, and the content of Coomassie brilliant blue R-250 is preferably 0.12-0.125%.
Another object of the present invention provides the higher modified coomassie brilliant blue staining method of a kind of sensitivity.
Coomassie brilliant blue staining method provided by the present invention may further comprise the steps:
1) SDS-PAGE glue is placed above-mentioned gel stationary liquid, following fixedly 0.5-2.0 hour of normal temperature;
2) glue is transferred in the acetum that concentration expressed in percentage by volume is 0.5-3.0% under the normal temperature extension 1.0-3.0 hour;
3) glue is transferred in the above-mentioned coomassie brilliant blue staining agent, normal temperature dyeed 8-24 hour down;
4) glue is transferred among the destainer I, vibration decolouring 1-3 hour, transfer to continue to decolour among the destainer II to background clean till; Described destainer I is that to contain concentration expressed in percentage by volume be 4-6% acetic acid, the aqueous solution of 30-50% ethanol; Described destainer II is that to contain concentration expressed in percentage by volume be 2-4% acetic acid, the aqueous solution of 20-40% ethanol.
In above-mentioned colouring method, the set time in the step 1) is preferably 1 hour.
Step 2) concentration expressed in percentage by volume of acetum is preferably 1.0% in, and the extension time is preferably 2 hours.
SDS-PAGE glue is preferably 2 hours at the bleaching time of destainer I in the step 4); Acetic acid content among the described destainer I is preferably 5%, ethanol content preferred 40%; Acetic acid content among the described destainer II is preferably 3%, and ethanol content is preferably 30%; Described vibration velocity is preferably 50-100rpm.
The invention provides a kind of coomassie brilliant blue staining method and special gel immobile liquid and coloring agent.Compare with conventional gel stationary liquid, gel stationary liquid of the present invention has added concentration expressed in percentage by volume 8-12% methyl alcohol, makes proteopexy more firm, has avoided losing of small molecular weight protein as much as possible.Compare with conventional coomassie brilliant blue staining agent, the mass percentage concentration of Coomassie brilliant blue is brought up to 0.1-0.15% in the coomassie brilliant blue staining agent of the present invention, makes Color more obvious.In addition, compare with the method for dying of examining of routine, the method for dying of examining of the present invention has increased the extension step, makes glue expand, and it is big that factor of porosity becomes, and is beneficial to dyestuff better and the protein combination in the glue.By unidirectional and coloured differently effect dielectrophoresis glue are relatively found, it is sensitiveer more than Blue Silver method that the present invention examines the method for dying, can be at the protein that detects on the nanogram level level on the SDS-PAGE glue, and much simpler than the operation of argentation, have conveniently advantage.Colouring method of the present invention and special gel immobile liquid thereof and coloring agent will play a significant role in the research of proteomics, have a extensive future.
Below in conjunction with specific embodiment the present invention is described in further detail.
Description of drawings
Fig. 1 is for silver dyes, Blue Silver dyes and the present invention examines the comparative result and the sensitivity testing result of examining the method for dying of the present invention of dying the method Color
Fig. 2 is for silver dyes, Blue Silver dyes and the present invention examines the Color comparative result of the method for dying to 2-DE glue
Embodiment
Method therefor is conventional method if no special instructions among the following embodiment.
1, the unidirectional electrophoresis detection of salicornia europaeal (Salicornia europaea L.) aerial part total protein
Gel stationary liquid: containing concentration expressed in percentage by volume is 10% acetic acid, 40% ethanol and 10% methanol in water.
The coomassie brilliant blue staining mother liquor: it is that vibration is 3 hours in 37 ℃ of shaking tables, is mixed with the coomassie brilliant blue staining mother liquor in 95% the ethanol that 2.5 gram Coomassie brilliant blue R-250 are dissolved in 1 liter of concentration expressed in percentage by volume, and room temperature preservation is filtered before using.
The coomassie brilliant blue staining agent: be made into the coomassie brilliant blue staining mother liquor that to contain concentration expressed in percentage by volume be 5% acetic acid, 45% ethanol and mass volume concentrations are the aqueous solution of 0.125% Coomassie brilliant blue R-250.
Destainer I: containing concentration expressed in percentage by volume is 5% acetic acid, the aqueous solution of 40% ethanol.
Destainer II: containing concentration expressed in percentage by volume is 3% acetic acid, the aqueous solution of 30% ethanol.
With 0 (contrast), 200,400,600 and 800mM NaCl salicornia europaeal (Salicornia europaea L.) is handled, adopt phenol to take out method (S.C.Carpentier, E.Witters, K.Laukens, P.Deckers, R.Swennen, B.Panis, Preparation of protein extracts from recalcitrant planttissues:An evaluation of different methods for two-dimensional gelelectrophoresis analysis, Proteomics 5 (2005) 2497-2507.) extract the total protein of the above-mentioned salicornia europaeal aerial part of handling through NaCl, then with the 2-DE electrophoresis apparatus of peace horse West Asia company and with reference to people such as instructions and Yan (J.X.Yan, W.Robin, B.Tom, A.H.Rachel, A.W.Jules, Westbrook, H.W.Colin, J.D.Michael, A modified silver staining protocol forvisualization of proteins compatible with matrix-assisted laserdesorption/ionization and electrospray ionization-mass spectrometry.Electrophoresis 21 (2000) 3666-3672.) method is carried out unidirectional electrophoresis detection, the applied sample amount of each swimming lane is 20 micrograms, adopt the coloured differently method that the protein on the SDS-PAGE glue is dyeed then, more final Color, argentation is undertaken by people's such as above-mentioned Yan method, Blue Silver decoration method is carried out (G.Candiano according to people's such as Candiano method, M.Bruschi, L.Musante, L.Santucci, G.M.Ghiggeri, B.Carnemolla, P.Orecchia, L.Zardi, P.G.Righetti, Blue silver:A verysensitive colloidal Coomassie G-250 staining for proteome analysis, Electrophoresis 25 (2004) 1327-1333.), carry out coomassie brilliant blue staining with method of the present invention, may further comprise the steps:
1) SDS-PAGE glue is placed above-mentioned gel stationary liquid, fix 1 hour under the normal temperature;
2) glue being transferred to concentration expressed in percentage by volume is in 1.0% the acetum, and normal temperature extended 2 hours down;
3) glue is transferred in the above-mentioned coomassie brilliant blue staining agent, normal temperature dyeed 12 hours down;
4) glue is transferred among the destainer I, vibration decolouring is 2 hours under 80rpm, transfer to destainer II relaying persistent oscillation decolour to background clean till.The clean glue that decolours can be preserved in 7% acetic acid 1-3 month.
(figure A: the present invention examines the coloration result of the method for dying, the coloration result of figure B:Blue Silver method, figure C: the coloration result of argentation as shown in Figure 1 to the Color of unidirectional running gel dyeing for three kinds of colouring methods; Swimming lane 1-5 is respectively through 0,200,400,600 and the salicornia europaeal aerial part total protein handled of 800mM NaCl, swimming lane M is Marker), it is clear that the present invention examines the adhesive tape band that dyes method dyeing, background very clean (seeing the A part among Fig. 1), and utilize people such as Candiano the dyeing of Blue Silver method glue (seeing B part among Fig. 1) and all show very dark dyeing background according to the glue (seeing C part among Fig. 1) that people's such as Yan argentation dyes.On all unidirectional glue, the numerous protein band is all arranged, distribution range has covered greater than 94kDa with less than the zone of 14kDa molecular weight, still, and on the glue of Blue Silver dyeing, many minor band, particularly be positioned at the less important band in low-molecular-weight zone, very difficult quilt dyes, though can detect nearly all protein band with argentation,, because it is too dark that silver dyes background, many minor band are difficult to make a distinction from so dark dyeing background.
2, the dielectrophoresis of salicornia europaeal (Salicornia europaea L.) aerial part total protein detects
With 200mM NaCl salicornia europaeal (Salicornia europaea L.) is handled, adopt the phenol method of taking out to extract the total protein of the above-mentioned salicornia europaeal aerial part of handling through NaCl, 2-DE electrophoresis apparatus with peace horse West Asia company carries out the dielectrophoresis detection then, utilize 24 centimetres of IPG adhesive tape of pH 3-10 to carry out isoelectric focusing earlier, every adhesive tape applied sample amount is about 700 micrograms, through second behind electrophoresis, to SDS-PAGE glue use that the method identical with step 1 carries out respectively that silver dyes, Blue Silver dyeing and the present invention examine dyeing, Color relatively.
(figure A: the present invention examines the coloration result of the method for dying as shown in Figure 2 to the Color of dielectrophoresis glue dyeing for three kinds of colouring methods, the coloration result of figure B:Blue Silver method, figure C: the coloration result of argentation), on the whole, examine the 2-DE glue that dyes method dyeing with the present invention and have more protein site than 2-DE glue with the dyeing of Blue Silver method, Color is also far better, reaches the effect that silver dyes basically.It should be noted that through the present invention and examine the glue that dyes method dyeing, background is very clean.Choose with representative zone on the 2-DE glue of above-mentioned three kinds of distinct methods dyeing and (mark with square frame: a, b, c, d and e) analyze, on the 2-DE glue protein site count out and focusing quality on many difference are all arranged, in selected zone, have many protein sites to examine the method for dying by the present invention at an easy rate or argentation detects, but on glue, do not detect (seeing the regional c among the figure two, d and e) with Blue Silver dyeing.In addition, also from examine the 2-DE glue that dyes method dyeing with the present invention, choose more than 100 protein site and carried out mass spectrum evaluation and database retrieval, qualification result shows that colouring method of the present invention also has good mass spectrum compatibility, can be used for proteomics research fully.
The sensitivity that embodiment 2, the present invention examine the method for dying detects
The testing result of embodiment 1 has tentatively shown the superiority that the present invention examines the method for dying, now by unidirectional electrophoresis, as standard specimen further detection is done in the sensitivity that the present invention examines the method for dying with bovine serum albumin(BSA) (BSA), applied sample amount is respectively 10 μ g, 5 μ g, 3 μ g, 1 μ g, 0.5 μ g, 0.3 μ g, 0.1 μ g, 80ng, 50ng, 30ng, 10ng, 8ng and 5ng, testing result is seen figure D among Fig. 1, and (swimming lane 1-13 is followed successively by 10 μ g, 5 μ g, 3 μ g, 1 μ g, 0.5 μ g, 0.3 μ g, 0.1 μ g, 80ng, 50ng, 30ng, 10ng, the BSA of 8ng and 5ng applied sample amount, swimming lane M is Marker), drop to 10 nanograms at total protein concentration and (be equivalent to 1ng/mm
2) time, molecular weight is about the protein master tape of 62kDa in this albumen, can also be detected (swimming lane 11), shows that the method for dying of examining of the present invention has higher sensitivity, can detect protein on nanogram level level.
Claims (9)
1, the gel stationary liquid that is used for coomassie brilliant blue staining is that to contain concentration expressed in percentage by volume be 8-12% acetic acid, 30-50% ethanol and 5-15% methanol in water.
2, gel stationary liquid according to claim 1 is characterized in that: the volumn concentration of described acetic acid is 10%, and the volumn concentration of ethanol is 40%, and the methyl alcohol volumn concentration is 10%.
3, coomassie brilliant blue staining agent is that to contain concentration expressed in percentage by volume be 4-6% acetic acid, and 35-55% ethanol and mass volume concentrations are the aqueous solution of 0.1-0.15% Coomassie brilliant blue R-250.
4, coloring agent according to claim 3 is characterized in that: the volumn concentration of described acetic acid is 5%, and the volumn concentration of ethanol is 45%, and the mass volume concentrations of Coomassie brilliant blue R-250 is 0.12-0.125%.
5, a kind of coomassie brilliant blue staining method may further comprise the steps:
1) SDS-PAGE glue is placed claim 1 or 2 described gel stationary liquids, following fixedly 0.5-2.0 hour of normal temperature;
2) glue is transferred in the acetum that concentration expressed in percentage by volume is 0.5-3.0% under the normal temperature extension 1.0-3.0 hour;
3) glue is transferred in claim 3 or the 4 described coomassie brilliant blue staining agent, normal temperature dyeed 8-24 hour down;
4) glue is transferred among the destainer I, vibration decolouring 1-3 hour, transfer to continue to decolour among the destainer II clean to background; Described destainer I is that to contain concentration expressed in percentage by volume be 4-6% acetic acid, the aqueous solution of 30-50% ethanol; Described destainer II is that to contain concentration expressed in percentage by volume be 2-4% acetic acid, the aqueous solution of 20-40% ethanol.
6, method according to claim 5 is characterized in that: the set time in the described step 1) is 1 hour.
7, method according to claim 5 is characterized in that: the concentration expressed in percentage by volume of acetum is 1.0% described step 2), and the extension time is 2 hours.
8, method according to claim 5 is characterized in that: SDS-PAGE glue is 2 hours at the bleaching time of destainer I in the described step 4); The volumn concentration of acetic acid is 5% among the described destainer I, and the volumn concentration of ethanol is 40%; The volumn concentration of acetic acid is 3% among the described destainer II, and the volumn concentration of ethanol is 30%; Described vibration velocity is 50-100rpm.
9. the application of each described method of claim 5-8 in the qualitative and/or detection by quantitative of protein.
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| CN101871858A (en) * | 2010-06-22 | 2010-10-27 | 中国烟草总公司郑州烟草研究院 | SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) rapid decoloring method and special decoloring cup thereof |
| CN102288470A (en) * | 2011-07-20 | 2011-12-21 | 中国热带农业科学院热带生物技术研究所 | Coomassie brilliant blue G250 staining method, special staining solution and application thereof |
| CN104004382A (en) * | 2014-05-20 | 2014-08-27 | 北京五康新兴科技有限公司 | Coomassie brilliant blue staining solution and staining method |
| CN105445073A (en) * | 2015-09-14 | 2016-03-30 | 青岛爱迪生物科技有限公司 | Rapid protein staining solution |
| CN109520804A (en) * | 2018-11-20 | 2019-03-26 | 王雨璇 | A kind of quick coomassie brilliant blue staining liquid |
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| DE60108630T2 (en) * | 2000-06-02 | 2006-03-30 | BioControl Systems, Inc., Bellevue | INDEPENDENT DEVICES FOR DETECTING BIOLOGICAL CONTAMINANTS |
| CN1262834C (en) * | 2004-09-29 | 2006-07-05 | 中国人民解放军第二军医大学 | Method for increasing resolution power and signal strength in protein gel electrolytic zone |
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| CN101871858A (en) * | 2010-06-22 | 2010-10-27 | 中国烟草总公司郑州烟草研究院 | SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) rapid decoloring method and special decoloring cup thereof |
| CN102288470A (en) * | 2011-07-20 | 2011-12-21 | 中国热带农业科学院热带生物技术研究所 | Coomassie brilliant blue G250 staining method, special staining solution and application thereof |
| CN104004382A (en) * | 2014-05-20 | 2014-08-27 | 北京五康新兴科技有限公司 | Coomassie brilliant blue staining solution and staining method |
| 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 |
| CN105445073A (en) * | 2015-09-14 | 2016-03-30 | 青岛爱迪生物科技有限公司 | Rapid protein staining solution |
| CN105445073B (en) * | 2015-09-14 | 2018-09-25 | 青岛爱迪生物科技有限公司 | A kind of fast protein dyeing liquor |
| CN109520804A (en) * | 2018-11-20 | 2019-03-26 | 王雨璇 | A kind of quick coomassie brilliant blue staining liquid |
| CN109520804B (en) * | 2018-11-20 | 2019-09-24 | 王雨璇 | A kind of quick coomassie brilliant blue staining liquid |
| CN109632434A (en) * | 2018-12-25 | 2019-04-16 | 苏州译酶生物科技有限公司 | The Coomassie brilliant blue rapid dye liquor and dyeing-decolorzing method of a kind of low stimulation of low toxicity |
| CN109632434B (en) * | 2018-12-25 | 2021-03-26 | 苏州译酶生物科技有限公司 | Low-toxicity and low-irritation Coomassie brilliant blue rapid dyeing liquid and dyeing and decoloring method |
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