CN103308370B - Single blue A is as the purposes of protein pre-dyed toner - Google Patents
Single blue A is as the purposes of protein pre-dyed toner Download PDFInfo
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Abstract
The invention discloses single blue A as the purposes of protein pre-dyed toner, belong to protein staining field.The present invention finds, adopt single blue A as the pre-dyed toner of protein, have the advantages such as staining procedure is easy, dyeing time is short, coloring agent consumption is few, the protein product after dyeing can naked eyes visual inspection also can adopt scanner to detect under near-infrared excitation light.When dyeing to protein with single blue A, adopt base catalysis or electric shock catalysis can promote staining reaction, improve staining efficiency, the present invention is optimized each technological parameter of base catalysis or electric shock catalysis further.Is the present invention preparing protein molecular weight standard product, is optimizing Western-blot, In-Gel? have wide practical use in location in cell of the detection mode of Western or tube polyacrylamide gel electrophoresis and object observing albumen or distribution situation.
Description
Technical field
The present invention relates to the novelty teabag of single blue A, particularly relate to the novelty teabag of single blue A as protein pre-dyed toner, belong to the application of single blue A.
Background technology
Proteomics (proteomics) refers to all proteins of genome encoding for research object, the composition of Study on Protein and Changing Pattern thereof from cellular level and integral level, thus is deeply familiar with organic various physiology and pathology.Compare with traditional protein research, proteomics research embodies comprehensive, globality, high flux, on a large scale feature.The research of proteomics is carried out proof analysis for the Leaf proteins of the theoretical prediction completing genome plan and is had irreplaceable vital role, the more important thing is that Proteomic analysis is without the need to relying on genome research, utilize protein research to be expected to find the biological function clue of reluctant " orphan " gene without any homologous sequence of sequential analysis from protein expression rule, and then disclose its status at whole functional network.Proteomic techniques is comparatively complicated, comprises the content of Separation of Proteins, qualification and information analysis three aspect.Wherein, gel electrophoresis is one of core technology corresponding with separation andpreconcentration.
Use polyacrylamide as the supporting dielectric of electrophoresis first from nineteen fifty-nine Raymond and Weintraub, since particularly early 1960s, Hjerten, Ornstein and Davis delivered the element task of discontinuous electro-phoresis system, polyacrylamide gel has become the most frequently used supporting dielectric of current biochemical test, has been become the common method of Separation of Proteins, analysis by the SDS-PAGE technology using strong anion SDS to set up.And in proteomics experiment, the dyeing of protein after polyacrylamide gel electrophoresis is a very important link.Conventional protein staining method has coomassie brilliant blue staining, silver dye, fluorescent dye etc.Wherein, Coomassie brilliant blue (CBBR and CBBG) dyeing is the most frequently used protein staining method, and it has, and cost is low, simple operation and the feature such as mass spectrum compatibility is good.But the sensitivity of coomassie brilliant blue staining overall dye is still lower, be about hundreds of nanogram to a few microgram, dyeing time is longer, and gel background is partially dark.Silver dye is the protein detection method the sensitiveest except radioactive label of generally acknowledging at present, can detect albumen in nanogram level level.But silver dye has relatively low repeatability usually, causes very high background, and can modify protein due to the glutaraldehyde that adds and formaldehyde, cause the compatible performance of the subsequent protein group research of silver dye generally not good.Fluorescence dye since latest developments has very high sensitivity and subsequent protein group research compatibility.But because fluorescent dye is expensive, cut very easily cancellation, also need high-end instrument and equipment and special analysis software, limit the widespread use of this technology at proteomics field.
Western-blot gel electrophoresis is one of follow-up proteomics research technology, and it is mainly used for identifying, quantize and determine the size of specific protein.Western-blot is developed by Northern-blot and Southern-blot.In 20 century 70 later stages, the people such as Towbin (1979), use polyacrylamide-urea gel electrophoresis isolated protein, and transfer on nitrocellulose filter.Burnette(1981) use widely used SDS-PAGE (SDS-PAGE) isolated protein, this finally result in this method and is called as western blot.It is also referred to as Western blotting or Western blotting, and becomes rapidly the powerful of proteomics research.The method of Western-blot first uses the protein of gel electrophoresis separating natural or sex change, then protein transduction is moved on on film, with unlabelled specificity primary antibodie with transfer to the antigen on film and be combined, add two of mark marks such as (radiation) element, biotins again to resist and carry out hybridization check, have that detecting step is many, high in cost of production defect, have much room for improvement.
Summary of the invention
One of the object of the invention is to provide the novelty teabag of single blue A as protein pre-dyed toner;
Two of the object of the invention is optimized by the method for single blue A staining for protein;
Three of the object of the invention there is provided a kind of protein detection method of improvement;
Four of object of the present invention is to provide a kind of method detecting albumen distribution situation in cell.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
For the various defects existing for existing protein pre-dyed toner or colouring method, present inventor has performed and study with keen determination, final discovery, single blue A can as the pre-dyed toner of protein, with the pre-dyed toner of single blue A as protein, staining procedure is easy, dyeing required time is short, the amount that can complete dyestuff needed for dyeing, dyeing in 15 minutes is few, protein product after dyeing can naked eyes visual inspection also can be detected by special scanners device under near-infrared excitation light, thus completes the present invention.
The present invention provide firstly the novelty teabag of single blue A as protein pre-dyed toner; Single blue A can have multiple use in the fields such as the mark of protein or detection, such as: will make the destination protein screening and identification etc. of pre-dyed protein molecular weight standard product (marker) and mass spectrophotometry front end after single for the protein of standard volume blue A dyeing.
Further, the invention provides method protein dyeed with single blue A, the method comprises: single blue solution A mixed with protein solution to be dyed and carry out staining reaction, adds after dyeing optimization agent room temperature leaves standstill and adds dye-terminators termination staining reaction again.
Wherein, the volume ratio of single blue A and protein to be dyed is preferably 1:9.
The protein being applicable to single blue A dyeing can be that any one needs to adopt the method for dyeing to carry out the protein of qualitative or quantitative detection, such as: as the protein of protein molecular weight standard product (marker) in Protein Detection; Can be the protein as primary antibodie in Western-blot or In-GelWestern detection method; Can also be the various protein etc. for observing at intracellular targeting or distribution.
Thus, the invention provides a kind of prestained protein molecular weight standard product, by preparing after single for the protein of standard volume blue A dyeing.
The invention provides a kind of Western-blot method of detection protein of optimization, the method comprises: be separated protein to be detected with gel electrophoresis; The protein transduction that gel electrophoresis is separated is moved on on film; By after single for primary antibodie blue A dye marker with transfer to the antigen protein on film and be combined; Directly detect under infrared excitation light.
The method combines after single for primary antibodie blue A dyeing with the protein on film, by infrared detection system, primary antibodie to be directly detected down infrared, eliminate and isotope or biotin labeled two anti-steps of carrying out hybridization check, significantly shorten detection time, reduce testing cost.
In addition, in In-GelWestern analyzes, carry out hybridization check by after target protein or the single blue A dyeing of primary antibodie, not only greatly reduce the time needed for experiment and cost, also there is good signal to noise ratio (S/N ratio).
The present invention finds further, via the target protein of single blue A mark, also can be combined with infrared detection system and be used for object observing albumen in situations such as intracellular distribution or location; Therefore, the invention provides a kind of method that object observing albumen distributes in cell, comprising: by after single for target protein blue A dye marker with cell incubation, adopt infrared detection system object observing albumen in intracellular location or distribution situation
The present invention is found further by test, and when carrying out staining reaction after single blue solution A mix with protein solution, the mode of employing base catalysis, electric shock or heating is carried out catalysis and is conducive to raising staining efficiency.
Thus, the invention provides a kind of method improving protein staining efficiency, comprise: after the blue solution A of list being mixed with protein solution, under the condition of base catalysis, shock by electricity catalysis or heatable catalytic, carry out catalysis staining reaction, adding after dyeing optimization agent room temperature leaves standstill and adding dye-terminators termination staining reaction again.
Wherein, described alkali is preferably weak base, is more preferably sodium bicarbonate solution; The present invention finds, when carrying out staining reaction, the quality ratio of the alkali added and protein to be dyed has appreciable impact for staining efficiency, when the mass ratio of sodium bicarbonate and protein is 1:8-1:13, Color is best, especially, when the mass ratio of sodium bicarbonate and protein is 1:12, there is best Color.
When the mode catalysis staining reaction by electric shock, the magnitude of voltage adopted and electric shock time all directly have influence on the height of staining efficiency; The present invention is found by test, can raising protein staining efficiency in various degree when adopting the voltage of 600-900V to carry out catalysis, wherein, when voltage is 900V, and Color when protein staining effect is better than other voltage from entirety; Particularly, when voltage is 900V, when the electric shock time was more than 20 seconds, protein staining efficiency has obvious lifting, and especially when adopting the voltage of 900V to shock by electricity, when the electric shock time reaches 25 seconds, protein staining effect is far superior to the Color of other electric shock time; But, when the electric shock time more than 25 seconds time, protein staining effect sharply glides again.Therefore, when with single blue A preparation of dyestuff pre-dyed albumen, it is 900V that the condition of electro-catalysis is preferably voltage, and the electric shock time is preferably 20-25 second, most preferably is 25 seconds.
Described heatable catalytic mode is preferably 100 DEG C, mixed sample heating 1min; Add 100 μ l dyeing subsequently and optimize agent and 100 DEG C of heating 1min.
To sum up, single blue A is adopted to have cheap as protein staining agent, simple to operate, dyeing required time is short, sensitivity is moderate, observe simple and easy, gel background is very shallow, the advantages such as compatibility is good of subsequent protein group research, can have application prospect comparatively widely preparing in location in cell of protein molecular weight standard product (marker), the detection mode of optimization Western-blot, In-GelWestern or tube polyacrylamide gel electrophoresis and object observing albumen or distribution situation.
Accompanying drawing explanation
The molecular structural formula of the mono-blue A of Fig. 1.
10% polyacrylamide gel electrophoresis qualification that the blue A of Fig. 2 base catalysis list dyes and the blue A of electro-catalysis list dyes: object band is bovine serum albumin(BSA) (BSA), and size is 60KD.1: the blue A dyeing of electro-catalysis list; 2: the blue A dyeing of base catalysis list; M: pre-dyed standard items molecular weight protein.
10% polyacrylamide gel electrophoresis qualification after bovine serum albumin(BSA) (BSA) purifying of the blue A mark of Fig. 3 thermocatalysis list; Object stripe size is about 60KD.M: pre-dyed standard molecular weight albumen.
The standard molecular weight self-control pre-dyed protein 15 % polyacrylamide gel electrophoresis qualification of the mono-blue A pre-dyed of Fig. 4 and coomassie brilliant blue staining: object stripe size is distributed as 96KD, 60KD, 36KD and 19KD; Fig. 4 A: the standard molecular weight self-control pre-dyed albumen of single blue A pre-dyed; Fig. 4 B: coomassie brilliant blue staining after standard molecular weight self-control protein electrophoresis; M: pre-dyed standard molecular weight albumen.
The blue A of Fig. 5 complex samples list dyes 10% polyacrylamide gel electrophoresis qualification; Experimental group is respectively NAMPT protein crude extract administration and NAMPT purifying protein; M: pre-dyed standard molecular weight albumen.
Bovine serum albumin(BSA) (BSA) the gradient qualification of the mono-blue A mark of Fig. 6; Object band is 60KD.M: pre-dyed standard molecular weight albumen; 1:1 μ gBSA albumen; 2:0.1 μ gBSA albumen; 3:0.01 μ gBSA albumen.
The Optimal Experimental result of the base catalyzed reactions of the mono-blue A preparation of dyestuff pre-dyed albumen of Fig. 7.
The Optimal Experimental result of the electric shock catalytic reaction of the mono-blue A preparation of dyestuff pre-dyed albumen of Fig. 8.
15% polyacrylamide gel electrophoresis qualification after the non-specific IgG antibody purifying of the mono-blue A mark of Fig. 9; Object stripe size is about 160KD; Fig. 9 A: observations under naked eyes; Fig. 9 B:Odyssey infrared thermoviewer imaging results.M: pre-dyed standard molecular weight albumen.
The In-GelWestern result of the non-specific IgG antibody of the mono-blue A mark of Figure 10; Redness is single blue A dye signal, the green goat anti-rabbit igg hybridization signal for IRDye800CW mark; M: pre-dyed standard molecular weight albumen.
Figure 11 tube polyacrylamide gel electrophoresis; Object stripe size is respectively 80KD and 50KD.
The bovine serum albumin(BSA) cell endocytic laser co-focusing qualification of the mono-blue A mark of Figure 12; Blue is nucleus, the red bovine serum albumin(BSA) for single blue A mark.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
1. experiment material:
1.1 dyestuff
Single blue A dyestuff is purchased from SIGMA company.
1.2 albumen and antibody
Bovine serum albumin(BSA) (BSA) is purchased from Genview company, and the non-specific IgG antibody in rabbit source is purchased from Abcam company of Britain.Taq enzyme albumen is purchased from precious bioengineering (Dalian) company limited.NAMPT albumen is provided by the prompt teacher of this laboratory oil red.
2. experiment reagent
Tris salt: the modern east fine chemicals company limited in Beijing
DTT(dithiothreitol (DTT)): Beijing is glad through biotechnology Ltd of section
IAA(iodoacetamide): SIGMA company of the U.S.
Sodium bicarbonate: Xi'an chemical reagent factory
Glucosan G-50: Long March pharmaceutical factory of Shanghai pharmaceuticals
SDS(lauryl sodium sulfate): Shanghai Sheng Gong bioengineering company limited
EDTA(ethylenediamine tetraacetic acid): Shanghai Sheng Gong bioengineering company limited
Acrylamide: methene acrylamide (29:1): Beijing Ding Guo biotechnology Ltd
APS(ammonium persulfate): Beijing is glad through biotechnology Ltd of section
TEMED(N, N, N', N'-tetramethylethylenediamine): Beijing is glad through biotechnology Ltd of section
Glycocoll: Beijing Yili Fine Chemicals Co., Ltd.
Pre-dyed standard molecular weight albumen: NEB company of the U.S.
Isopropyl alcohol: the modern east fine chemicals company limited in Beijing
Acetic acid: the modern east fine chemicals company limited in Beijing
Sodium chloride: the modern east fine chemicals company limited in Beijing
Potassium chloride: Shanghai Sheng Gong bioengineering company limited
Potassium dihydrogen phosphate: Shanghai Sheng Gong bioengineering company limited
Sodium hydrogen phosphate: Beijing Yili Fine Chemicals Co., Ltd.
Tween-20: SIGMA company of the U.S.
The goat anti-rabbit igg of IRDye800CW mark: LI-COR company of the U.S.
Coomassie brilliant blue R_250: Beijing Ding Guo biotechnology Ltd
Absolute ethyl alcohol: the modern east fine chemicals company limited in Beijing
3. the preparation of main agents
The required reagent of 3.1 dyeing
single blue A dyestuff
Take the mono-blue A dyestuff of 0.1g, 0.01g sodium bicarbonate, deionized water dissolving is also settled to 1ml, 4 DEG C of preservations.
agent is optimized in dyeing
Take 0.003g dithiothreitol (DTT), 0.0242gTris salt, after deionized water dissolving, then add 0.1ml glycerine, deionized water is settled to 1ml, 4 DEG C of preservations.
dye-terminators
Take 0.1g iodoacetamide, deionized water dissolving is also settled to 1ml, 4 DEG C of preservations.
3.2SDS-PAGE electrophoresis:
10%APS
Take 0.1g ammonium persulfate, deionized water dissolving is also settled to 1ml, 4 DEG C of preservations, uses in 1 week.
10%SDS
Take 5gSDS, deionized water dissolving is also settled to 40ml and is heated to 68 DEG C of hydrotropies, and enriching HCl adjusts pH5 to 7.2, adds dried uply to be settled to 50ml.
1.5MTris·HCl（pH8.8）
Take 45.43gTris salt, after deionized water dissolving, by dense HCl adjust ph to 8.8, be finally settled to 250ml with deionized water, 4 DEG C of preservations.
1.0MTris·HCl（pH6.8）
10 take 30.29gTris salt, after deionized water dissolving, regulate pH to 6.8, be finally settled to 250ml with deionized water with dense HCl.4 DEG C of preservations.
10 × electrophoretic buffer
Take 30.5gTris salt, 144.8g glycocoll, 10gSDS, deionized water is settled to 1000ml.
Reagent needed for 3.3In-GelWestern:
immobile liquid
Measure 500ml isopropyl alcohol, 50ml acetic acid, deionized water is settled to 1000ml.
1ｘPBS（0.05M，pH7.4）
Take Na
2hPO
412H
2o3.58g, KH
2pO
42H
2o0.24g, NaCl8g, KCl0.2g are dissolved in 1000ml deionized water, 1MHCl adjust ph to 7.4, place, be stored in 4 DEG C after cooling after autoclaving in super-clean bench.
Reagent needed for 3.4 coomassie brilliant blue stainings:
fixing dyeing liquor
Take 2.5g coomassie brilliant blue R_250 respectively, measure 225ml ethanol, 50ml acetic acid, deionized water is settled to 500ml.
destainer
Measure 225ml ethanol, 50ml acetic acid, deionized water is settled to 500ml.
The embodiment 1 various colouring method of single blue A staining for protein and the rear protein purification of dyeing
1. by the method that single blue A dyes to protein
1.1 heatable catalytic staining reactions
(1) get the mono-blue A dyeing liquor of 10 μ l fully to mix with 90 μ l protein solutions.
(2) by 100 DEG C, mixed sample heating 1min.
(3) add 100 μ l dyeing subsequently and optimize agent, and 100 DEG C of heating 1min.
(4) treat that sample is cooled to room temperature, add 20 μ l dye-terminators, room temperature leaves standstill 5min.
1.2 electro photoluminescence catalysis staining reactions
(1) get the mono-blue A dyeing liquor of 10 μ l fully to mix with 90 μ l10%BSA protein solutions.
(2) mixed sample is added 200ml electricity revolving cup.
(3) electroporation selects 1800V as output voltage, electric shock 20ms.
(4) add 100 μ l dyeing subsequently and optimize agent, room temperature leaves standstill 5min.
(5) add 20 μ l dye-terminators, room temperature leaves standstill 5min.
1.3 base catalysis staining reactions
(1) get the mono-blue A dyeing liquor of 10 μ l fully to mix with 90 μ lBSA protein solutions.
(2) add 20 μ l1M sodium bicarbonates, room temperature leaves standstill 5min.
(3) add 100 μ l dyeing subsequently and optimize agent, room temperature leaves standstill 5min.
(4) add 20 μ l dye-terminators, room temperature leaves standstill 5min.
2. protein purification and qualification after dyeing
Protein purification after 2.1 dyeing
(1) take 5g glucosan G-50, deionized water is settled to 50ml, boils 3h with boiling water.
(2) gel stirred loading is tied in the 0.5mlEP pipe in hole in bottom in advance.
(3) the 0.5mlEP pipe that gel is housed is placed in a 1.5mlEP pipe.
(4) subsequently the protein solution after dyeing is added in 0.5mlEP pipe.
(5) eluent is collected.
2.2SDS-PAGE electroresis appraisal
(1) glass plate thick for cleaned 1.0mm is fixed on encapsulating frame.
(2) prepare the resolving polyacrylamide gel of 10%, full dose application of sample is in glass plate, and slowly add 2ml absolute ethyl alcohol, ambient temperatare puts 30min.
(3) after lower gelling is solid, outwell absolute ethyl alcohol, add 2ml polyacrylamide and concentrate glue, plug comb at Jiao Mianshang simultaneously.
(4) after gluing is solidified, slowly pull out comb, application of sample is in loading hole, and the leakage of electricity of 80V voltage is swum, and until sample when the interface of concentrated glue and separation gel pressure is into a line, is about 30min, adjusts voltage to 140V.
(5) separately two pieces of glass plates, the concentrated glue of excision, naked eyes or with the imaging of Odyssey infrared thermoviewer.
Base catalysis, electric shock catalysis and the blue A of the thermocatalysis list 10% polyacrylamide gel electrophoresis qualification result that dyes is shown in Fig. 2 and Fig. 3 respectively.From experimental result, with when singly blue A is as protein staining agent, adopt base catalysis, electro-catalysis or thermocatalytic mode all can promote protein staining.
Experimental example 1 single blue A preparation of dyestuff pre-dyed albumen marker and qualification
1 experimental technique
1.1marker protein mixes
Get 0.1 μ gTaq zymoprotein, BSA albumen, lactate dehydrogenase A albumen and IL-7 respectively to add in 1.5mlEP pipe, fully mix, obtain undyed mixed protein molecular weight standards albumen (marker protein).
1.2marker protein staining
(1) add the mono-blue A dyestuff of 3 μ l, fully mix.
(2) by 100 DEG C, mixed sample heating 1min.
(3) add 30 μ l dyeing subsequently and optimize agent, and 100 DEG C of heating 1min.
(4) treat that sample is cooled to room temperature, add 0.6 μ l dye-terminators, room temperature leaves standstill 5min.
1.3marker protein purification
(1) take 5g glucosan G-50, deionized water is settled to 50ml, boils 3h with boiling water.
(2) gel stirred loading is tied in the 0.5mlEP pipe in hole in bottom in advance.
(3) the 0.5mlEP pipe that gel is housed is placed in a 1.5mlEP pipe.
(4) subsequently the marker protein solution after dyeing is added in 0.5mlEP pipe.
(5) eluent is collected.
The qualification of 1.4 pre-dsred protein marker
(1) glass plate thick for cleaned 1.0mm is fixed on encapsulating frame.
(2) prepare the resolving polyacrylamide gel of 10%, full dose application of sample is in glass plate, and slowly add 2ml absolute ethyl alcohol, ambient temperatare puts 30min.
(3) after lower gelling is solid, outwell absolute ethyl alcohol, add 2ml polyacrylamide and concentrate glue, plug comb at Jiao Mianshang simultaneously.
(4) after gluing is solidified, slowly pull out comb, add the marker protein of 20 μ l pre-dyed in loading hole, the leakage of electricity of 80V voltage is swum, and until sample when the interface of concentrated glue and separation gel pressure is into a line, is about 30min, adjusts voltage to 140V.
(5) two pieces of glass plates are separated, the concentrated glue of excision, visual inspection protein band.
(6) gel is put into coomassie brilliant blue staining liquid 10min.Period constantly shakes.
(7) gel is put into destainer, repeatedly change destainer till gel background purifies.
2, experimental result
Carry out pre-dyed by above method to marker protein, the design of experimental group is the marker protein using coomassie brilliant blue staining after using the marker protein of single blue A dyestuff pre-dyed and electrophoresis respectively.Experimental result shows, can by naked eyes identification and band consistent with the band after coomassie brilliant blue staining (Fig. 4, Fig. 5) by the protein marker of single blue A colouring method pre-dyed.Experimental result shows that single blue A protein staining method may be used for the preparation of protein marker.
The single blue A of experimental example 2 marks bovine serum albumin gradient test experience
1, experimental technique
Get 3 1.5mlEP pipes respectively, add BSA protein 10 μ l, 1 μ l, 0.1 μ l that concentration is 1 μ g/ μ l respectively, then to adding the distilled water adding 9 μ l in EP pipe that 1 μ l concentration is the BSA albumen of 1 μ g/ μ l, to add 0.1 μ l concentration 1 μ g/ μ l BSA albumen EP pipe in add the distilled water of 9.9 μ l, fully mix.Take out 1 μ l solution by every pipe, and add the mono-blue A dyestuff (200mM) of 1 μ l respectively, 100 DEG C of heating 1min.Treat that sample cools, often pipe adds 10 μ l dyeing optimization agent respectively, 100 DEG C of heating 1min.Treat that 3 pipes are cooled to room temperature, add 2 μ l dye-terminators respectively, room temperature leaves standstill 5min.Carry out SDS-PAGE electroresis appraisal subsequently.
2, experimental result
By the imaging of Odyssey infrared thermoviewer, visible 3 pipe samples all present clear band, and band is tapered with the reduction of protein concentration, and when protein concentration is 0.01 μ g/ μ l, band is superfine, not easily distinguishes (Fig. 6).Experimental result shows, and the Cmin of single blue A dye marker protein is 0.01 μ g/ μ l.
The Optimal Experimental of the base catalyzed reactions of the single blue A preparation of dyestuff pre-dyed albumen of experimental example 3
One, experimental technique
1. get 17 1.5mlEP pipes and add 9 μ lBSA protein solutions (concentration is 1 μ g/ μ l) respectively, then add the mono-blue A dyestuff (200mM) of 1 μ l respectively in every pipe; In every pipe, (at random) adds 4 μ l, 5 μ l, 6 μ l, 7 μ l, 8 μ l, 9 μ l, 10 μ l, 11 μ l, 12 μ l, 13 μ l, 14 μ l, 15 μ l, 16 μ l, 17 μ l, 18 μ l, 19 μ l and 20 μ l sodium bicarbonate solutions (concentration is 1.12mol/L) respectively again, mix, room temperature leaves standstill 5min.
2. in pipe, add 10 μ l dyeing more respectively optimize agent, room temperature leaves standstill 5min.
3. add 2 μ l dye-terminators in the most backward pipe, room temperature leaves standstill 5min.
4. the sample in 5 pipes is added respectively in 5 holes in 96 orifice plates.
5. arrange Biotec microplate reader parameter, exciting light is 640nm, 96 orifice plates is put into measurement and draws reading.
Two, experimental result
Experimental result is in table 1 and Fig. 7.
The Optimal Experimental result of table 1 base catalyzed reactions
From experimental result, when the addition of sodium bicarbonate solution is between 7 μ l-12 μ l, testing result has higher OD value, and Color is better; When the consumption of sodium bicarbonate solution is 8 μ l, OD value reaches the highest (0.361), along with sodium bicarbonate solution consumption more than 8 μ l time, OD value declines rapidly, and Color sharply declines; In other words, when the mass ratio of sodium bicarbonate and protein to be dyed is 1:8 to 1:13, especially 1:11.968(is about 1:12) time, there is best Color.
The Optimal Experimental of the electrocatalytic reaction of the single blue A preparation of dyestuff pre-dyed albumen of experimental example 4
One, experimental technique
1. in electric revolving cup, add 360 μ lBSA protein solutions (concentration is 1 μ g/ μ l), then add the mono-blue A dyestuff (200mM) of 40 μ l wherein; Electric revolving cup is put into eppendorf electroporation.
2. setting electroporation parameter is 600V, 5s, shocks by electricity.
3. when 10s, take out electric revolving cup, get 10 μ l and put into 1.5mlEP pipe.Again electric revolving cup is put into electroporation, continue electric shock 5s, namely take out during 15s, get 10 μ l and put into a new 1.5EP pipe.
4. repeat 3 steps to 25s.
5. setting electroporation parameter is 800V, 5s, shocks by electricity.
6. repeat step 3,4;
7. setting electroporation parameter is 900V, 5s, shocks by electricity.
8. repeat step 3,4;
9. the 12 pipe samples collected are added respectively 10 μ l dyeing and optimize agent, room temperature leaves standstill 5min.
10. in pipe, add 2 μ l dye-terminators again, room temperature leaves standstill 5min.
Sample in 12 pipes to add in 12 holes in 96 orifice plates by 11. respectively.
12. arrange bio-tec microplate reader parameter, and exciting light is 640nm, 96 orifice plates is put into measurement and draws reading.
Two, experimental result
Testing result (OD
640) in table 2 and Fig. 8.
The Optimal Experimental result of table 2 electrocatalytic reaction
From experimental result, when voltage is 900V, Color when protein staining effect is better than other voltage from entirety; Particularly, when voltage is 900V, when the electric shock time was more than 20 seconds, OD value promotes significantly, and when the voltage electric shock time of 900V reaches 25 seconds, OD value reaches mxm. (0.238), far away higher than the OD value of other electric shock time; When the electric shock time more than 25 seconds time, OD value is again in sharply downtrending.Therefore, when with single blue A preparation of dyestuff pre-dyed albumen, it is 900V that the condition of electro-catalysis is preferably voltage, and the electric shock time is preferably 20-25 second, most preferably is 25 seconds.
The experiment of In-GelWestern detection mode optimized by the single blue A dyestuff of experimental example 5
One uses the non-specific IgG antibody of single blue A dye marker
1, experimental technique
Get the mono-blue A dyeing liquor of 10 μ l fully to mix with the non-specific IgG antibody solution of 90 μ l; By 100 DEG C, mixed sample heating 1min.Add 100 μ l dyeing subsequently and optimize agent, and 100 DEG C of heating 1min.Treat that sample is cooled to room temperature, add 20 μ l dye-terminators, room temperature leaves standstill 5min.Take 5g glucosan G-50, deionized water is settled to 50ml, boils 3h with boiling water.The gel stirred is loaded and ties in the 0.5mlEP pipe in hole in bottom in advance.The 0.5mlEP pipe that gel is housed is placed in a 1.5mlEP pipe.Subsequently the non-specific IgG antibody solution after dyeing is added in pipe.Collect eluent.Carry out SDS-PAGE electroresis appraisal subsequently.
Specific experiment group is in table 3.
Table 3
2, experimental result
Experimental result is presented at the visible clear obvious IgG antibody protein band (Fig. 9) of about 160KD.Result shows, and single blue A colouring method may be used for the dye marker of antibody.
Two, In-GelWestern is carried out by the non-specific IgG antibody of single blue A dye marker
1, experimental technique
Gel after electrophoresis is put into immobile liquid and hatches 15min.Remove immobile liquid, wash 15min with deionized water, and slowly shake.In the 5%BSA solution suitable containing 0.1%Tween-20, press the dilution proportion two anti-(goat anti-rabbit igg of IRDye800CW mark) of 1:1000, slowly shake gel is hatched two anti-4 DEG C spend the night, lucifuge operates.Slowly shake at enough PBS+0.1%Tween-20 and point wash glue 10min 3 times.Glue 5min is washed in PBS.With the imaging of Odyssey infrared thermoviewer.Specific experiment group is in table 1.
2, experimental result
The non-specific IgG antibody of the single blue A mark of experimental result display can be with markd two anti-specific bindings, and with undressed non-specific IgG antibody by the band of the anti-mark of specificity two consistent (Figure 10).Experimental result display may be used for Western-blot through the antibody of single blue A pre-dyed.
Experimental example 6tube polyacrylamide gel electrophoresis
1, experimental technique
Get one section of 20cm flexible pipe, pour into 10% resolving polyacrylamide gel prepared wherein, at one end stay the loading hole of 2cm.Treat that gelling is solid, in loading hole, add the NAMPT albumen after the purifying of the mono-blue A mark of 3 μ l, and close loading hole with 10% polyacrylamide gel solidified prepared.After placing tube, 150V constant voltage 1h30min, close electrophoresis apparatus.Tube is taken out with the imaging of Odyssey infrared thermoviewer.
2, experimental result
By the imaging of Odyssey infrared thermoviewer, two corresponding object bands (Figure 11) clearly can be seen in pipe.Result shows, and the albumen of single blue A mark can carry out simple and rapid Isolation and Identification with tube electrophoresis.
The albumen of the single blue A mark of experimental example 7 is in intracellular test experience
1, experimental technique
Sterile cover slips is preset in 12 orifice plates, after HeLa plating cells 24h, is containing the viviparous cow's serum DMEM of 10%, 37 DEG C of 5%CO
2be cultured to 90% to converge.After adding the bovine serum albumin(BSA) 2h of single blue A mark, 3 times are cleaned with PBS, 15min is fixed with 4% paraformaldehyde, through the abundant rinsing of PBS, with the fluorescence mountant mounting containing DAPI, imaging is carried out at blue light and infrared excitation passage respectively under being placed in LeicaDM4000B laser confocal microscope, and through pseudo-colours superposition synthesis storage figure picture.
2, experimental result
The nucleus of experimental result visible blue and the BSA albumen (Figure 12) of the blue A mark of coverlet that is all at core and intracytoplasmic redness.The results show, the protein of single blue A mark can be detected in cell.
Claims (4)
1. single blue A is as the purposes of protein pre-dyed toner; Comprise: after single blue solution A being mixed with the protein that needs dye, under the condition shocked by electricity, carry out staining reaction; The voltage of described electric shock is 900V; The described electric shock time is 25 seconds;
Add after agent is optimized in dyeing and add dye-terminators cessation reaction again.
2. according to purposes according to claim 1, it is characterized in that, described protein comprises protein molecular weight standard thing, antibody, the destination protein of mass spectrophotometry front end or the foreign protein of expressing in cell or tissue.
3. according to purposes according to claim 2, it is characterized in that: described antibody is primary antibodie used during Western-blot or In-GelWestern detects.
4. according to purposes according to claim 1, it is characterized in that: the volume ratio of single blue A and protein is 1:9.
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|---|---|---|---|---|
| WO2011071359A2 (en) * | 2009-12-09 | 2011-06-16 | Instituto Tecnológico y de Estudios Superiores de Monterrey | Covalent modification of proteins for the instantaneous visualisation thereof and subsequent characterisation through mass spectrometry |
| CN201917487U (en) * | 2010-12-31 | 2011-08-03 | 南京金斯瑞生物科技有限公司 | Instrument used for quick protein dying and transfer printing |
| CN102443046A (en) * | 2011-11-09 | 2012-05-09 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 | Method for efficiently extracting sheep viscus tissue protein |
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|---|---|---|---|---|
| WO2011071359A2 (en) * | 2009-12-09 | 2011-06-16 | Instituto Tecnológico y de Estudios Superiores de Monterrey | Covalent modification of proteins for the instantaneous visualisation thereof and subsequent characterisation through mass spectrometry |
| CN201917487U (en) * | 2010-12-31 | 2011-08-03 | 南京金斯瑞生物科技有限公司 | Instrument used for quick protein dying and transfer printing |
| CN102443046A (en) * | 2011-11-09 | 2012-05-09 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 | Method for efficiently extracting sheep viscus tissue protein |
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| Accelerated Identification of Proteins by Mass Spectrometry by Employing Covalent Pre-Gel Staining with Uniblue A;Marco A. et al.;《PLoS one》;20120229;第7卷(第2期);第7-8页 * |
| Lysine-directed staining of proteins for MS-based analyses;Matthew T. et al.;《Electrophoresis》;20130328;第34卷(第3期);第401-404页 * |
| 红外荧光素Alexa Fluor 647标记的2型腺相关病毒保持天然的感染活性;贾书勤等;《中国生物化学与分子生物学报》;20081231;第24卷(第12期);第1154页右栏第1段，第1155页左栏第3段 * |
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