蛋白質(zhì)－臨床博士

1、蛋白質(zhì)研究的技術(shù)與策略蛋白質(zhì)研究的技術(shù)與策略任惠民任惠民自報(bào)家門（任惠民）自報(bào)家門（任惠民）u畢業(yè)：華東師范大學(xué)畢業(yè)：華東師范大學(xué) 化學(xué)系化學(xué)系u原工作單位：中國(guó)科學(xué)院上海生理研究所原工作單位：中國(guó)科學(xué)院上海生理研究所u97年引進(jìn)至年引進(jìn)至 （原）上海醫(yī)科大學(xué)神經(jīng)病（原）上海醫(yī)科大學(xué)神經(jīng)病學(xué)研究所學(xué)研究所u國(guó)外工作：日本杏林大學(xué)，美國(guó)加州大學(xué)國(guó)外工作：日本杏林大學(xué)，美國(guó)加州大學(xué)u承擔(dān)基金：國(guó)家、省部、中科院、重點(diǎn)實(shí)承擔(dān)基金：國(guó)家、省部、中科院、重點(diǎn)實(shí)驗(yàn)室等驗(yàn)室等u發(fā)表論著：發(fā)表論著：180余篇余篇研究蛋白質(zhì)的意義研究蛋白質(zhì)的意義 蛋白質(zhì)的重要性蛋白質(zhì)的重要性 構(gòu)成生命體的最重要物質(zhì)構(gòu)成生命體的

2、最重要物質(zhì) 與生命活動(dòng)緊密相關(guān)與生命活動(dòng)緊密相關(guān) 蛋白質(zhì)與疾病的關(guān)系蛋白質(zhì)與疾病的關(guān)系 表達(dá)水平與功能改變導(dǎo)致疾病發(fā)生表達(dá)水平與功能改變導(dǎo)致疾病發(fā)生內(nèi)內(nèi) 容容 蛋白質(zhì)研究的前處理技術(shù)蛋白質(zhì)研究的前處理技術(shù) 蛋白質(zhì)的分離技術(shù)蛋白質(zhì)的分離技術(shù) 蛋白質(zhì)的凝膠電泳技術(shù)蛋白質(zhì)的凝膠電泳技術(shù)western blotwestern blot （蛋白免疫印跡）（蛋白免疫印跡） 蛋白質(zhì)組學(xué)蛋白質(zhì)組學(xué)proteomics 蛋白質(zhì)的凝膠電泳技術(shù)電泳（電泳（electrophoresis）電泳是指帶電粒子在電場(chǎng)中向與自身帶相反電荷的電泳是指帶電粒子在電場(chǎng)中向與自身帶相反電荷的電極移動(dòng)的現(xiàn)象。電極移動(dòng)的現(xiàn)象。 （1 1

3、）類型）類型 液相：在液相介質(zhì)中液相：在液相介質(zhì)中 固相：固相中或固相表面固相：固相中或固相表面 （2 2）凝膠電泳）凝膠電泳 水平：瓊脂電泳、瓊脂糖電泳等水平：瓊脂電泳、瓊脂糖電泳等 垂直：垂直： 聚丙烯酰胺凝膠電泳聚丙烯酰胺凝膠電泳用用 途途 提供蛋白質(zhì)分子量、電荷、亞基結(jié)構(gòu)、分離純化提供蛋白質(zhì)分子量、電荷、亞基結(jié)構(gòu)、分離純化 蛋白質(zhì)的純度；蛋白質(zhì)的純度； 對(duì)蛋白復(fù)雜混合物的定性分析；對(duì)蛋白復(fù)雜混合物的定性分析； 了解不同生理病理時(shí)特定組織或細(xì)胞中蛋白質(zhì)的了解不同生理病理時(shí)特定組織或細(xì)胞中蛋白質(zhì)的 變化；變化； 適用范圍廣泛。適用范圍廣泛。 特特 點(diǎn)點(diǎn) 成本低：設(shè)備、人力、時(shí)間成本低：設(shè)備

4、、人力、時(shí)間 靈敏度高：可檢測(cè)靈敏度高：可檢測(cè)g、甚至、甚至ng水平的量水平的量 分辨率高：可分離數(shù)百、甚至上千種不同成分分辨率高：可分離數(shù)百、甚至上千種不同成分 重復(fù)性好：可重復(fù)重復(fù)性好：可重復(fù) 可信度高：罕有假象發(fā)生可信度高：罕有假象發(fā)生structure and properties of protein proteins - polymer of amino acids with biological activity made of alpha amino acid (20) structure of amino acids aas have a carboxyl group (-c

5、ooh) & amino group (-nh2) and are often ionized at physiological pheffect of ph and buffer on effect of ph and buffer on protein chargeprotein charge proteins are amphoteric compounds and are therefore either positively or negatively charged depending on the ph of their local enviroment post tra

6、nslation modifications the addition of charged and uncharged sugar the addition of phosphate group sulphydryl cross-link isoelectric point - ph where there is no net charge in moleculemigration of protein depends onmigration of protein depends on strength of electric field (heat) net charge on molec

7、ule (ph) size and shape of molecules (choice of support) properties of supporting medium (viscosity, electroendosmosis)蛋白質(zhì)電泳類型蛋白質(zhì)電泳類型 按材質(zhì)按材質(zhì) 濾紙電泳、淀粉膠電泳、醋酸薄膜電泳、濾紙電泳、淀粉膠電泳、醋酸薄膜電泳、 瓊脂電泳、瓊脂糖電泳、瓊脂電泳、瓊脂糖電泳、 聚丙烯酰胺凝膠電泳、毛細(xì)管電泳聚丙烯酰胺凝膠電泳、毛細(xì)管電泳 按形狀按形狀 圓盤（圓盤（disc）、平板（）、平板（slab）l按電泳條件按電泳條件 恒壓、恒流、常壓、高壓恒壓、恒流、常壓、高壓 聚

8、丙烯酰胺凝膠電泳聚丙烯酰胺凝膠電泳polyacrylamide gel electrophoresis 變性電泳：變性電泳：sodium dodesyl sulphate-plyacrylamide gel electrophoresis (sds-page) 非變性電泳：非變性電泳：native (buffer) gels 等點(diǎn)聚焦：等點(diǎn)聚焦：isoelectric focusing (ief) gels 雙向電泳：雙向電泳： two dimension polyacrylamide gel electrophoresis (2d-gels) 凝膠制作凝膠制作原理原理丙烯酰胺丙烯酰胺 單體單

9、體甲叉雙丙烯酰胺甲叉雙丙烯酰胺 交聯(lián)劑交聯(lián)劑過(guò)硫酸氨過(guò)硫酸氨 加速劑加速劑四甲基乙二胺四甲基乙二胺 催化劑催化劑native gel electrophoresis for the detection of particular proteins (i.e., an enzyme) on the basis of its biological activity polyacrylamide (normally 7.5% gel), but the sds is absent and the proteins are not denatured prior loading proteins se

10、parate according to their different electrophoretic mobilities and the sieving effect （篩孔效應(yīng)）（篩孔效應(yīng)）of the gel the enzyme of interest can be identified by incubating the gel in an appropriates substratesds-page sds-page (sds-polyacrylamide gel electrophoresis)(sds-polyacrylamide gel electrophoresis) s

11、eparate protein according to size samples must be previously boiled 5 minutes in sample buffer containing: sds (ch3-(ch2)10-ch2oso3sds (ch3-(ch2)10-ch2oso3- - nana+ +), ), 1 molecule binds every 2 amino acids residues1 molecule binds every 2 amino acids residues -mercaptoethanol-mercaptoethanol（ - -

12、巰基乙醇巰基乙醇 ） sucrose or glycerolsucrose or glycerol ionizable tracking dye (i.e., bromophenol blue)ionizable tracking dye (i.e., bromophenol blue)sample buffer (laemmli 2x buffer) 4% sds、10% -mercaptoehtanol、 20% glycerol 0.004% bromophenol blue in 0.125 m tris hclsds-page the original charge on prote

13、in is masked by the negatively charged of sdssds-pagesds-page 連續(xù)連續(xù) 非連續(xù)非連續(xù)ph 8.8 gelph 8.8 gelph 8.8 gelph 8.8 gel，分離膠分離膠ph 6.8 gel, ph 6.8 gel, 濃縮膠濃縮膠適用于提取的樣品蛋白濃適用于提取的樣品蛋白濃度較高，加樣體積較少。度較高，加樣體積較少。適用于提取樣品蛋白濃度適用于提取樣品蛋白濃度較低，需要加樣體積較大較低，需要加樣體積較大sds-pagesds-page 凝膠配制：凝膠配制： 30 g 丙烯酰胺、丙烯酰胺、1 g bis-丙烯酰、丙烯酰、1 g

14、 sds 溶于水至溶于水至100 ml。 分離膠緩沖液（分離膠緩沖液（tris-cl, ph 8.8）：）： 7.2 g tris、1 g sds 溶于水，鹽酸調(diào)溶于水，鹽酸調(diào)ph=8.8，加水至，加水至100 ml。 濃縮膠緩沖液（濃縮膠緩沖液（tris-cl, ph 6.8）：）： 3 g tris、 0.1 g sds 溶于水，鹽酸調(diào)溶于水，鹽酸調(diào)ph=6.8，加水至，加水至100 ml。 電泳緩沖液：電泳緩沖液： 28.8 g甘氨酸、甘氨酸、6 g tris、 1 g sds 溶于水至溶于水至1000 ml。recommended acrylamide concentration re

15、commended acrylamide concentration for protein electrophoresisfor protein electrophoresisseparation size range (kd) % acrylamide 36-205 5% 24-205 7.5% 14-205 10% 14-66 12.5% 14-45 15%the larger proteins fail to move significantly into the gelstrategy：changing cross-linking density（交聯(lián)度）（交聯(lián)度）sds-page

16、and cbb r-250 staininggradient gelsgradient gels the acrylamide concentration is varied uniformly from, typically 5% at the top of the gel to 25% acrylamide at the bottom of the gel5% 25%advantagesgreater range of protein mw values can be separated than on a fixed-percentage gelthe proteins with a v

17、ery similar mw values may be resolvedsds-page常見(jiàn)問(wèn)題與解決方案常見(jiàn)問(wèn)題與解決方案l膠不平？膠不平？l凝膠漏液？凝膠漏液？n膠板洗刷干凈膠板洗刷干凈n加入加入aps和和temed的量要合適。的量要合適。n加入試劑后搖勻，使其充分混加入試劑后搖勻，使其充分混 合，防止部分膠塊聚合不均勻。合，防止部分膠塊聚合不均勻。n溫度合適，受熱不均勻?qū)е履z聚溫度合適，受熱不均勻?qū)е履z聚 合不均勻。合不均勻。n兩塊玻璃板底部要對(duì)齊。兩塊玻璃板底部要對(duì)齊。sds-page常見(jiàn)問(wèn)題與解決方案常見(jiàn)問(wèn)題與解決方案p條帶比正常的條帶比正常的 窄？窄？p“微笑微笑”或或“倒倒 微

18、笑微笑”條帶？條帶？l凝膠聚合不均勻，灌膠時(shí)候盡凝膠聚合不均勻，灌膠時(shí)候盡 量混合均勻，動(dòng)作輕緩。量混合均勻，動(dòng)作輕緩。l拔梳子要迅速，清洗加樣孔要拔梳子要迅速，清洗加樣孔要 小心，以免把上樣帶扭曲。小心，以免把上樣帶扭曲。l樣品鹽濃度過(guò)高會(huì)擠壓其他條樣品鹽濃度過(guò)高會(huì)擠壓其他條 帶導(dǎo)致寬窄不一，純化樣品，帶導(dǎo)致寬窄不一，純化樣品， 調(diào)整鹽濃度。調(diào)整鹽濃度。l膠板底部有氣泡會(huì)影響電泳效膠板底部有氣泡會(huì)影響電泳效 果，應(yīng)趕走氣泡。同時(shí)注意電果，應(yīng)趕走氣泡。同時(shí)注意電 泳槽裝置是否合適。泳槽裝置是否合適。ife ife (isoelectric focusing electrophoresis)(i

19、soelectric focusing electrophoresis) separates proteins by isoelectric points large pore size of gel and equilibrium conditions minimize molecular sieving native or denaturing conditions possible generates ph gradient in electric field gradient range depends on ampholyte pka values anode: dilute aci

20、d (h3po4) cathode: dilute alkali (naoh)ifeife2d-page2d-page (two-dimension polyacrylamide gel electrophoresis) combines of ief (separating according to charge, pi) with size separation of sds-page1) 1) ife in narrow tubein the presence of 8m ureaand non-ionic detergent2) extruded from tube 2) extrud

21、ed from tube incubate in buffer containing incubate in buffer containing sds (15 min)sds (15 min)3) resolved in sds-pag gel3) resolved in sds-pag gel2d-page2d-page蛋白質(zhì)的染色蛋白質(zhì)的染色 （1）總蛋白的染色方法總蛋白的染色方法 正染：陽(yáng)離子染料正染：陽(yáng)離子染料 (如考馬斯亮蘭，如考馬斯亮蘭，coomassie brilliant blue) 負(fù)染：金屬陰離子負(fù)染：金屬陰離子 (如咪唑鋅，如咪唑鋅，imidazole-zinc) 銀染

22、：硝酸銀銀染：硝酸銀 其他：熒光染色或標(biāo)記，放射標(biāo)記（其他：熒光染色或標(biāo)記，放射標(biāo)記（fluorescent staining or labeling, and radiolabeling） （2）特殊染色方法特殊染色方法 糖基化蛋白糖基化蛋白 磷酸化蛋白磷酸化蛋白 selected staining methodsselected staining methods“classical” coomassie brilliant blue r-250 stain coomassie brilliant blue r-250 stain cbb r-250 staining solution :

23、cbb r-250 staining solution : 0.1% (w/v) cbb r-250 dye in 40% ethanol and 10% acetic acid. cbb r-250 destaining solution :cbb r-250 destaining solution : 40% (v/v) ethanol and 10% (v/v) acetic acid. coomassie r-250 : reddish tint coomassie g-250 : greenish tint detection limit 10100 ng fazekas de st

24、 groth, et al. biochim. biophys. acta 1963, 71:377391.“classical” coomassie brilliant blue r-250 staining protocol1. place the gel into a staining dish and fix the proteins for 30 min in 20% (w/v) trichloroacetic acid (tca,) with gentle shaking.2. the gel briefly (12 min) with cbb r-250 destaining s

25、olution (40% ethanol and 10% acetic acid) to remove excess tca.3. immerse the gel in cbb r-250 staining solution andshake for at least 3 h. staining can continue overnight if more convenient.4. b rinse the gel with deionized water 5. destain the gel with cbb r-250 destaining solution with shaking un

26、til the background is clear.6. for higher sensitivity and decreased background staining, immerse the gel in 1% (v/v) acetic acid.colloidal coomassie brilliant blue g-250 stain colloidal coomassie brilliant blue g-250 stain cbb g-250 staining solution :cbb g-250 staining solution : 0.12% (w/v) cbb g-

27、250 dye, 10% ammonium sulfate, 10% phosphoric acid, and 20% methanol.cbb-g-250 destaining solution :cbb-g-250 destaining solution : 5% (w/v) ammonium sulfate and 10% (v/v) methanol. in general, colloidal cbb g staining is regarded as more sensitive than cbb r in solvent solutions neuhoff v , et al.

28、electrophoresis .1988, 9 :255 262 . candiano g, et al. electrophoresis. 2004, 25:1327 1333 .colloidal coomassie brilliant blue g-250 staining protocol1. place the gel in 500 ml fixing solution (40% ethanol and 10% acetic acid) for 3 h. 2. wash the gel briefly (2 30 s) with deionized water.3. immerse

29、 the gel in colloidal cbb g-250 staining solution for at least 3-4 h. staining can continue overnight if more convenient.4. rinse the gel briefly (2 30 s) with deionized water. 5. incubate the gel with cbb g-250 destaining solution (5% ammonium sulfate and 10% methanol) for 20 min, further 20-min in

30、cubation in cbb g-250 destaining solution imidazole-zinc reverse stainimidazole-zinc reverse stain imidazole solution : imidazole solution : 0.2 m imidazole containing 0.1 % sds. dissolve 6.81 g imidazole and 500 mg sodium dodecyl sulfate (sds) in 500 ml water.zinc sulfate solution : zinc sulfate so

31、lution : 0.2 m zinc sulfate. dissolve 28.76 g zinc sulfate heptahydrate in 500 ml water. it is also possible to prepare concentrated (10) imidazole and zinc sulfate stock solutions110 ng protein/band is detected in sds-page gelsreverse-stained proteins can be efficiently eluted and used in biologica

32、l and enzymatic assays. fernandez-patron c ,et al. biotechniques.1992 12 :564 573 . castellanos-serra l and hardy e. nat. protoc. 2006, 1 :1544 1551imidazole-zinc reverse staining protocol 1. briefly rinse the gel (2 30 s) in freshly prepared milli q water2. incubate the gel in 500 ml 0.2 m imidazol

33、e containing 0.1% sds for 15 min.3. rinse the gel briefly (30 s) in water to remove excess imidazole solution.4. add 0.2 m zinc sulfate solution while manually shaking for about 1 min. observe the gel over a black surface. the gel background will become white and protein spots transparent. note: pro

34、longed incubation in the zn 2+ solution may result in diminished sensitivity or even complete loss of the image.5. immediately discard the zinc sulfate solution and wash the gel briefly (three times, 60 s each). keep the negatively stained gel in water at 4c until usesilver nitrate stainsilver nitra

35、te stainfixing solution :fixing solution : 40% (w/v) ethanol and 10% (w/v) acetic acidsensitizer :sensitizer : 0.02% sodium thiosulfate-pentahydrate (the solution must be prepared the day of use)silver nitrate solution :silver nitrate solution : 0.2% silver nitrate and 0.02% formaldehyde(37%).develo

36、per :developer : 3% sodium carbonate, 0.05% formaldehyde (37%), and 0.0005% sodium thiosulfate pentahydrate.stop solution :stop solution : 0.5% glycine. 100 pg to 1 ng protein can be detected blum h, et al. electrophoresis,1987,8 : 93 99 .silver nitrate stainsilver nitrate stain today, more than 100

37、 different variants of silver-staining protocols exist for proteins separated in polyacrylamide gels. however, in general, there are two large categories: alkaline and acidic silver stains alkaline methods : work with a diamine complex of silver nitrate in a highly alkaline environment (ammonia and

38、sodium hydroxide). patterns are then developed in dilute acidic solutions of formaldehyde. acidic methods: use silver nitrate in water (weakly acidic solutions) for gel impregnation and a development step in formaldehyde solutions at alkaline ph fast silver nitrate staining protocol 1. place the gel

39、 in 40% ethanol and 10% acetic acid at least for 3 h. 2. wash the gel in 30% ethanol for 20 min, followed by two consecutive washes (20 min each) with 15% ethanol and milli q water, respectively.3. sensitize with sodium thiosulfate solution for 1 min4. rinse the gel in water for 3 20 s while manuall

40、y shaking.5. add silver nitrate solution and gently agitate for 30 min. protect the tray from bright light.6. rinse the gel in water for 3 20 s while manually shaking.fast silver nitrate staining protocol7. quickly add developing solution (3% sodium carbonate, 37% formaldehyde). sometimes, a gray or

41、 brown precipitate may form. develop until protein spots are clearly visible (515 min). the most intense spots will appear within a few minutes.8. stop development as soon as an adequate degree of staining has been achieved to avoid excessive background formation.9. rinse the gel briefly in deionize

42、d water, and immerse it in stop solution (0.5% glycine or, alternatively, 4% tris and 2% acetic acid) for 20 min . then, wash the gel (3 10 min) with milli q water .fluorescence-based protein detection methods for total protein staining two major approaches(1) covalent derivatization of proteins wit

43、h fluorophores prior to ief(2) postelectrophoretic protein staining by the intercalation of fluorophores into the sds micelles coating the proteins by the direct electrostatic interaction with the proteins. for pre-electrophoretic fluorescent these dyes are commercially available as cydyes, labeling

44、 are the cyanine-based dyes that react with lysyl or cysteinyl residues.for post-electrophoretic fluorescent these dyes are sypro ruby and deep purple stain sypro ruby total protein stainsypro ruby total protein stain fixing solution : 40% (v/v) ethanol and 10% (v/v) acetic acid.sypro ruby staining

45、solution : ready-to-use solution (commercial).destaining solution : 10% (v/v) ethanol and 7% (v/v) acetic acid. 110 ng may be detected, and staining is reversible. sypro ruby can be used for multiplexed staining, combination with fluorescence detection of glyco- or phosphoproteinssypro ruby staining

46、 protocol 1. place the gel into a high-density polypropylene tray (e.g., a food box). do not use a glass vessel! fix the proteins in 40% ethanol and 10% acetic acid for at least 3 h. 2. add 500 ml sypro ruby staining solution incubating for at least 3 h at room temperature or overnight. protect the

47、reagent from bright light. 3. wash the gel (3 10 min) in deionized water or, preferably, with 10% ethanol and 7% acetic acid to reduce speckling, 4. capture and save the image using an appropriate fluorescence imager. use the filter sets that match the excitation (maximum, 450 nm) and emission (maxi

48、mum, 610 nm) wavelength for sypro ruby. images may also be obtained by using a simple uv transilluminator. for labeling protein samples for digefor labeling protein samples for dige sds sample buffer : 1% (w/v) sds, 100 mm tris-hcl (ph 8.5).thiourea/urea/chaps buffer a : 2 m thiourea, 7 m urea, 4% c

49、haps, 30 mm tris-hcl (ph 8.5).thiourea/urea/chaps buffer b : 2 m thiourea, 7 m urea, 4% chaps.dithiothreitol (dtt) solution : dtt 50% (w/v).cydye stock solution : reconstitute the cydyes (cy2, cy3 and cy5 in dimethylformamide (dmf) to a stock solution of 1 mm the reconstituted dyes are stable at 20c

50、 for 6 monthsstop solution : 10 mm lysine.minimal (lys) labeling protein samples 1. extract proteins, preferably with sds-buffer. determine the protein concentration of the sample with a protein assay. dilute the sds extract with a four fold excess thiourea/urea/ chaps of buffer b (final sds concent

51、ration 0.2%). the protein concentration should be between 5 and 10 mg/ml. instead of using sds buffer, proteins may be directly solubilized in thiourea/urea/chaps buffer a.2. directly before labeling, dilute the cydye stock solution to a final concentration of 0.4 mm (400 pmol/l), e.g., mix 3 l of d

52、mf with 2 l of reconstituted cydye.3. 50 g of protein sample add 400 pmol (1 l) of cydye . cool on ice. important : (1) make sure the ph is 8.5. (2) keep the dyes and the samples in the dark and on ice!4. vortex and centrifuge at 10,000 g for 5 s.minimal (lys) labeling protein samples5. incubate the

53、 sample in the dark for 30 min on ice.6. add 1 l of 10 mm lysine to stop the labeling reaction.7. mix, and incubate in the dark for 10 min on ice.8. add 1 l of dtt solution and 1 l of pharmalyte 3 9. 10 per 50 l of sample solution. labeled samples can be stored for at least 3 months at 70c.9. run ie

54、f and sds-page. protect from light to minimize photobleaching of the fluorescence dyes.pro-q diamond phosphoprotein pro-q diamond phosphoprotein stainstainfixing solution : 40% (v/v) ethanol and 10% (v/v) acetic acid.pro-q diamond phosphoprotein stain : ready-to-use solution (molecular probes, eugen

55、e, or, usa).pro-q diamond phosphoprotein destaining solution : 25% acetonitrile and 50 mm sodium acetate/acetic acid, ph 4.0. pro-q diamond phosphoprotein staining protocol 1. place the gel into a highdensity polypropylene tray (e.g., food box). do neither use a glass vessel2. fix the proteins in 40

56、% ethanol and 10% acetic acid (or 50% methanol and 10% acetic acid) overnight. replacement of the fixing solution after 1 h is highly recommended. fixation is critical for specific staining, sds would result in very poor or no staining of phosphoproteins.3. wash the gel with milli q water ( 3x 20 mi

57、n per wash).4. incubate the gel with pro-q diamond phosphoprotein stain for 23 h protect the reagent from light.5. destain with destaining solution (25% acetonitrile and 50 mm sodium acetate/acetic acid, ph 4.0; 3x 30 min per wash).6. wash the gel 2 5 min with milli q water.7. continue with image ac

58、quisition. pro-q diamond stain has excitation/emission maxima of 555/580 nm a visible-light transilluminator, or (with reduced sensitivity) a 300 nm transilluminatorphosphoproteins phosphorylation is a reversible widely used by cells for activation/inhibition of specific pathways both in basic energ

59、y metabolism and in specialized signal transduction processes. phosphorylation results in a shift of the protein pi to more acidic value. immunochemicals specific for p-tyrosine, p-serine and p-threonine have been developed for the p-variants of individual proteins.other phosphoproteins stain method

60、smethyl green ( basic dye) an old procedure depends on the hydrolysis of the phosphoester linkage of phosphoserine(絲氨酸絲氨酸) and phosphothreonine (蘇氨酸蘇氨酸)(but not of phosphotyrosine) . insoluble calcium phosphate to insoluble nitrophospho-molybdate complex by treated with ammonium molybdate. cutting ja, r