CN106289921B - The application of 2 ', 7 '-dichlorofluoresceins and its derivative in Protein Detection - Google Patents
The application of 2 ', 7 '-dichlorofluoresceins and its derivative in Protein Detection Download PDFInfo
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- 238000002331 protein detection Methods 0.000 title description 4
- 238000010186 staining Methods 0.000 claims abstract description 55
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 39
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 28
- 238000001514 detection method Methods 0.000 claims abstract description 6
- 238000004043 dyeing Methods 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 10
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 238000001962 electrophoresis Methods 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 5
- 230000002441 reversible effect Effects 0.000 claims description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 3
- 150000003863 ammonium salts Chemical class 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- 159000000000 sodium salts Chemical group 0.000 claims description 3
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 3
- 229940038773 trisodium citrate Drugs 0.000 claims description 3
- VFNKZQNIXUFLBC-UHFFFAOYSA-N 2',7'-dichlorofluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(Cl)=C(O)C=C1OC1=C2C=C(Cl)C(O)=C1 VFNKZQNIXUFLBC-UHFFFAOYSA-N 0.000 abstract description 23
- 230000035945 sensitivity Effects 0.000 abstract description 14
- 238000001502 gel electrophoresis Methods 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 2
- 238000004949 mass spectrometry Methods 0.000 abstract 1
- ALDXNLXJHWOGNT-UHFFFAOYSA-N 1h-imidazole;zinc Chemical class [Zn].C1=CNC=N1 ALDXNLXJHWOGNT-UHFFFAOYSA-N 0.000 description 13
- 239000000499 gel Substances 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 210000004556 brain Anatomy 0.000 description 7
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 6
- 239000000975 dye Substances 0.000 description 6
- 238000001819 mass spectrum Methods 0.000 description 6
- 208000002109 Argyria Diseases 0.000 description 4
- 239000003292 glue Substances 0.000 description 4
- 238000011017 operating method Methods 0.000 description 4
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 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 3
- 238000011161 development Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 150000002460 imidazoles Chemical class 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000011701 zinc Substances 0.000 description 3
- 229910052725 zinc Inorganic materials 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 235000005979 Citrus limon Nutrition 0.000 description 2
- 108010058846 Ovalbumin Proteins 0.000 description 2
- 108010065081 Phosphorylase b Proteins 0.000 description 2
- 239000002390 adhesive tape Substances 0.000 description 2
- 102000005936 beta-Galactosidase Human genes 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000004040 coloring Methods 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 229940092253 ovalbumin Drugs 0.000 description 2
- 238000010183 spectrum analysis Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 2
- -1 2 ', 7 '-dichlorofluorescein anion Chemical class 0.000 description 1
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 244000248349 Citrus limon Species 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical compound N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
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- 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
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- 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
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Abstract
本发明涉及蛋白质负染检测技术，具体地说是2′，7′‑二氯荧光素及其衍生物在蛋白质负染检测中的应用。本发明还提供了应用2′，7′‑二氯荧光素进行蛋白负染检测的方法，包括步骤：蛋白质凝胶电泳结束后直接染色；接着显影。本发明具有灵敏度高、操作简单迅速、重现性好、线性关系好、质谱兼容性好、使用安全、成本低廉等优点，可较好适用于高通量蛋白质组学的研究。The invention relates to a protein negative staining detection technology, in particular to the application of 2', 7'-dichlorofluorescein and its derivatives in protein negative staining detection. The invention also provides a method for protein negative staining detection using 2', 7'-dichlorofluorescein, comprising the steps of: direct staining after protein gel electrophoresis; and then developing. The invention has the advantages of high sensitivity, simple and rapid operation, good reproducibility, good linear relationship, good mass spectrometry compatibility, safe use, low cost and the like, and can be better applied to the research of high-throughput proteomics.
Description
Technical field
The present invention relates to protein negative staining detection techniques, specifically, the negative staining dye that the one kind for being related to protein detection is new
Material.
Background technique
In proteomics research, gel protein staining technique is to be connected the pass of two dimensional electrophoresis and downstream mass spectral analysis
Key technology.Current advanced mass spectrograph can analyze the trace protein of pg rank.Conventional gel protein staining technique has
Coomassie brilliant blue staining, silver staining, fluorescent staining etc..Dying method with coomassie brilliant blue sensitivity is low, and argentation is complicated for operation, mass spectrum is simultaneous
Capacitive is poor, and fluorescence colour is expensive.These factors seriously constrain the development of high throughput protein group.Although closely
Many new staining techniques are developed over year, but there are still following one or several disadvantages: sensitivity is low, poor reproducibility, poison
Property is big, mass spectrum is poor, expensive, cumbersome etc..New colouring method is found to adapt to current advanced mass spectrum point
Analysis technology has become the task of top priority for the genome times afterwards comprehensively pushing proteomics development.2 ', 7 '-dichlorofluoresceins are gel egg
White dyeing provides a kind of quick, sensitive method, and sensitivity reaches 0.1ng/band, can compare favourably with silver staining.And 2 ',
7 '-dichlorofluorescein negative staining methods are coloured to gel background, the structure of protein are not decorated or destruction, are had
Good mass spectrum compensates for the defect of argentation mass spectrum difference.2 ', 7 '-dichlorofluorescein negative staining will well
It is connected two dimensional electrophoresis and downstream mass spectral analysis, pushes the fast development of proteomics.
Summary of the invention
The purpose of the present invention is to provide the application of 2 ', 7 '-dichlorofluoresceins and its derivative in protein detection.
Related compound of the present invention refer to 2 ', 7 '-dichlorofluorescein anion be the sodium salt of parent nucleus, sylvite and
Ammonium salt etc..
2 ', 7 '-dichlorofluorescein parent nucleus are as follows:
To achieve the purpose of the present invention, the present invention provides a kind of methods of protein on running gel negative staining, including such as
Lower step:
1) protein example after SDS-PAGE electrophoresis is placed in 5~20min of dyeing in dyeing liquor, wherein dyeing liquor is
2 ', 7 '-dichlorofluoresceins containing w/v 0.05~1% or derivatives thereof, 0.05%~0.5% sodium carbonate and press body
Methanol aqueous solution of the product than 30%~70%；Preferred dyeing time is 10min, preferred 2 ', 7 '-dichlorofluorescein and its is spread out
The concentration of biology is 0.3%, and preferred concentration of sodium carbonate is 0.1%, and preferred methanol concentration is 50%.
2) dyeing liquor is discarded, 1~5min of developing liquid developing is added, wherein developer solution is the lemon containing w/v 1.4%
Lemon acid and 1% trisodium citrate (pH=4), preferred developing time be 2min.
3) it detects, after gel-colored, can be placed under black background and directly visually observe；Or in Epson V700 scanner
Under, reverse scan Mode scans, sensitivity is higher.
Early-stage study shows that the technology has the following advantages:
1) high sensitivity: 2 ', 7 '-dichlorofluorescein negative staining high sensitivities are in hundreds times of dying method with coomassie brilliant blue, and silver
Dye method sensitivity is suitable；
2) easy to operate rapid: operating procedure is few, can complete in 12min；
3) it favorable reproducibility: is influenced by external conditions such as temperature, shaking table hunting frequencies small；
4) good reversibility: it is easy decoloration；
5) mass spectrum is good: since 2 ', 7 '-dichlorofluorescein negative staining are dyed to gel background, gel of getting along well
On protein combine, protein structure is entirely unaffected by, so can be with the products instrument highly compatible such as LC-MS；
6) using safe: using the low dyestuff of toxicity, improving the safety of experimental implementation, environmental pollution is small；
7) at low cost.
Detailed description of the invention:
The chemical structure of Fig. 12 ', 7 '-dichlorofluorescein of.
Fig. 2 one on SDS-PAGE glue, (A) 2 ', 7 '-dichlorofluorescein negative staining, (B) eosin negative staining, (C) penta
The comparison of dialdehyde argentation and the imidazoles negative staining sensitivity of (D) zinc.Eosin negative staining, glutaraldehyde argentation and zinc imidazoles negative staining
Recorded according to document；Use the standard protein (SDS6H2) of Sigma company for sample.Band 1,50ng；Band 2,25ng；Band 3,
12.8ng；Band 4,6.4ng；Band 5,3.2ng；Band 6,1.6ng；Band 7,0.8ng；Band 8,0.4ng；Band 9,0.2ng；Band 10,
0.1ng。
Fig. 3 one on SDS-PAGE glue, (A, E) 2 ', 7 '-dichlorofluorescein negative staining, (B, F) eosin negative staining,
The comparison of (C, G) glutaraldehyde argentation and the imidazoles negative staining sensitivity of (D, H) zinc.Separating sample is respectively e. coli total protein
(A-D) and mouse brain total protein (E-H).Sample times times dilution from left to right.
For Fig. 4 two on SDS-PAGE glue, (A) 2 ', 7 '-dichlorofluorescein negative staining and (B) zinc imidazoles negative staining are sensitive
The comparison of degree.Separate sample mouse brain total protein；IPG adhesive tape is 13 centimetres long, pH4-7；Resolving gel concentration is 11.4%；On sample
Sample amount is 100 micrograms/adhesive tape.
Fig. 5 compares one (A) 2 ', 7 '-dichlorofluorescein negative staining and (B) zinc imidazoles negative staining on SDS-PAGE glue
Examine the comparison of the range of linearity for the standard protein known.Standard protein is after dyeing, wherein 3 represent albumen β-
Galactosidase, phosphorylase b and ovalbumin draw to obtain standard protein after the analysis of Image Lab image software
The linearity curve of applied sample amount and band intensity.
Specific embodiment
Below with reference to specifically embodiment, the present invention is further explained.It should be appreciated that these embodiments are merely to illustrate
The present invention, and cannot limit the scope of the invention.
The dyeing of embodiment 12 ', 7 '-dichlorofluorescein negative staining
Fig. 1 is the chemical structural formula of 2 ', 7 '-dichlorofluoresceins.
The 2 ' of Fig. 2~5, the experiment of 7 '-dichlorofluorescein protein negative staining are carried out using following step:
1) gel after electrophoresis is placed in 0.3%2 ', 7 '-dichlorofluoresceins/0.1% sodium carbonate/50% methanol dyeing liquor
Middle dyeing 10min.
2) dyeing liquor is discarded, develop 2min in trisodium citrate (pH=4) developer solution of 1.4% citric acid/1%.
3) after gel-colored, under Epson V700 scanner, reverse scan Mode scans.
Protein staining is carried out with the sodium salt, sylvite, ammonium salt of 2 ', 7 '-dichlorofluoresceins respectively according to the method described above, as a result
Show obtain the testing result similar to 2 ', 7 '-dichlorofluoresceins with these derivatives.
Fig. 2 is to illustrate one on SDS-PAGE, pair of 2 ', 7 '-dichlorofluorescein negative stainings and other decoration method effects
Than using protein standard substance.
According to the method for embodiment 1, it is dyed using different methods, (A) 2 ', 7 '-dichlorofluorescein negative staining,
(B) comparison of eosin negative staining, (C) glutaraldehyde argentation and the imidazoles negative staining sensitivity of (D) zinc.Eosin negative staining, glutaraldehyde
Argentation and zinc imidazoles negative staining are recorded according to document；Use the standard protein (SDS6H2) of Sigma company for sample.Band
1,50ng；Band 2,25ng；Band 3,12.8ng；Band 4,6.4ng；Band 5,3.2ng；Band 6,1.6ng；Band 7,0.8ng；Band 8,0.4ng；
Band 9,0.2ng；Band 10,0.1ng.As a result as shown in Fig. 2, 2 ', 7 '-dichlorofluorescein negative staining sensitivity highests.
Fig. 3 is to illustrate one on SDS-PAGE, pair of 2 ', 7 '-dichlorofluorescein negative stainings and other decoration method effects
Than using E.coli total protein and mouse brain total protein.
The extracting method of E.coli total protein is as follows: taking E.coli bacterium solution, flora is collected by centrifugation in 12000rpm, is added
Thallus is resuspended in the phosphate buffer (pH=7.0) of 0.02mol/L.Utilize Ultrasonic Cell Disruptor smudge cells.18000rpm centrifugation
30min collects supernatant to get E.coli gross protein extracting solution (crude protein product).Mouse brain total protein extraction method is as follows:
Mice brain tissues are cleaned with 4 DEG C of PBS solution, are cleaned 3 times altogether.Tissue grinder is carried out with liquid nitrogen later, will be ground resulting
Powder is fitted into the EP pipe weighed in advance.Lysate (9.5M urea, 0.1DTT, 2%CHAPS, 0.8% are added into EP pipe
Pharmalyte pH3-10) extremely final concentration of 1mg/mL.Sample ultrasonic is crushed 3 times, 1 minute every time, after finishing
4 DEG C of high speed centrifugation 30min of 15000rpm, take supernatant.Above-mentioned E.coli and mouse brain total protein are divided through one to gel electrophoresis
From rear, respectively with (A) 2 ', 7 '-dichlorofluorescein negative staining of embodiment 1, (B) eosin negative staining, (C) glutaraldehyde argentation and
(D) zinc imidazoles negative staining is dyed, as a result as shown in Figure 3.2 ', 7 '-dichlorofluorescein negative stainings of display are a kind of operation letters
It is single, the method for high sensitivity.
Fig. 4 is to illustrate two on SDS-PAGE, 2 ', 7 '-dichlorofluorescein negative stainings and zinc imidazoles negative staining effect
Comparison, using being mouse brain total protein.
It is total to the mouse brain separated through two to gel electrophoresis with 2 ', 7 '-dichlorofluorescein negative stainings and zinc imidazoles negative staining
Albumen is dyed, as a result as shown in Figure 4.The result shows that 2 ', 7 '-dichlorofluoresceins are negative for the zinc imidazoles negative staining that compares
More protein spots can be detected in dye method, have higher sensitivity.
Fig. 5 illustrates that the linear dynamic range of 2 ', 7 '-dichlorofluorescein negative stainings and zinc imidazoles negative staining compares.
Three kinds of different molecular weights standard protein (β-galactosidase, phosphorylase b and
Ovalbumin it) is separated in 10% polyacrylamide gel, through (A) 2 ', 7 '-dichlorofluorescein negative staining；(B) zinc imidazoles is negative
Dye method, with the linearity curve of Image Lab image software analytical standard albumen applied sample amount and band intensity, to compare two kinds of dyes
The range of linearity of color method.Standard protein applied sample amount range shows 2 ', 7 '-dichlorofluorescein negative staining by Fig. 5 for 0.1~50ng
Method and zinc imidazoles negative staining have the consistent range of linearity.
Other colouring methods and pertinent literature in Fig. 2~5
Eosin negative staining operating method bibliography: Cong, W.T., Hwang, S.Y., Jin, L.T., He, H.Z.,
Choi, J.K., High-throughput negative detection of SDS-PAGE separated proteins
And its application for proteomics.Electrophoresis 2010,3,411420.
Glutaraldehyde argentation operating method bibliography: Heukeshoven, D.J., Dernick, R., Simplified
method for silver staining of proteins in polyacrylamide gels and the
Mechanism of silver staining.Electrophoresis 1985,6,103-112.
Zinc imidazoles negative staining operating method bibliography: Castellanos-Serra, L., Proenza, W., Huerta,
V., Moritz, R.L., Simpson, R.J., Proteome analysis of polyacrylamide gel-separated
proteins visualized by reversible negative staining using imidazole-zinc
Salts.Electrophoresis 1999,20,732-737.
Claims (4)
1. a kind of gel protein negative staining detection method comprising step:
1) protein example after SDS-PAGE electrophoresis is placed in 5~20min of dyeing in dyeing liquor, wherein dyeing liquor is containing weight
Measure 2 ', 7 '-dichlorofluoresceins or derivatives thereof of volume ratio 0.05~1%, 0.05%~0.5% sodium carbonate and by volume
30%~70% methanol aqueous solution；Wherein the derivative is sodium salt, sylvite or the ammonium salt of 2 ', 7 '-dichlorofluoresceins；
2) 1~5min of developing liquid developing is added, wherein developer solution is the citric acid and 1% containing w/v 1.4% of pH=4
Trisodium citrate；
3) it detects.
2. the method as described in claim 1, which is characterized in that dyeing time is 10min in step 1), and dyeing liquor is containing weight
2 ', 7 '-dichlorofluoresceins of volume ratio 0.3%, 0.1% sodium carbonate and 50% methanol aqueous solution by volume.
3. the method as described in claim 1, which is characterized in that developing time is 2min in step 2).
4. the method as described in claim 1, which is characterized in that step 3) is detected as with the naked eye directly observing, or uses Epson
V700 scanner reverse scan Mode scans.
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| CN102590100B (en) * | 2011-01-18 | 2016-08-31 | 温州安得森生物科技有限公司 | The application in protein detection of eosin and derivant thereof |
| US8945865B2 (en) * | 2011-03-10 | 2015-02-03 | Eisai R&D Management Co., Ltd. | Method for screening for compound capable of enhancing or inhibiting OATP1B1 transport activity, and method for determining expression level of OATP1B1 |
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