蛋白质－临床博士

1、蛋白质研究的技术与策略蛋白质研究的技术与策略任惠民任惠民自报家门（任惠民）自报家门（任惠民）u毕业：华东师范大学毕业：华东师范大学 化学系化学系u原工作单位：中国科学院上海生理研究所原工作单位：中国科学院上海生理研究所u97年引进至年引进至 （原）上海医科大学神经病（原）上海医科大学神经病学研究所学研究所u国外工作：日本杏林大学，美国加州大学国外工作：日本杏林大学，美国加州大学u承担基金：国家、省部、中科院、重点实承担基金：国家、省部、中科院、重点实验室等验室等u发表论著：发表论著：180余篇余篇研究蛋白质的意义研究蛋白质的意义 蛋白质的重要性蛋白质的重要性 构成生命体的最重要物质构成生命体的

2、最重要物质 与生命活动紧密相关与生命活动紧密相关 蛋白质与疾病的关系蛋白质与疾病的关系 表达水平与功能改变导致疾病发生表达水平与功能改变导致疾病发生内内 容容 蛋白质研究的前处理技术蛋白质研究的前处理技术 蛋白质的分离技术蛋白质的分离技术 蛋白质的凝胶电泳技术蛋白质的凝胶电泳技术western blotwestern blot （蛋白免疫印迹）（蛋白免疫印迹） 蛋白质组学蛋白质组学proteomics 蛋白质的凝胶电泳技术电泳（电泳（electrophoresis）电泳是指带电粒子在电场中向与自身带相反电荷的电泳是指带电粒子在电场中向与自身带相反电荷的电极移动的现象。电极移动的现象。 （1 1

3、）类型）类型 液相：在液相介质中液相：在液相介质中 固相：固相中或固相表面固相：固相中或固相表面 （2 2）凝胶电泳）凝胶电泳 水平：琼脂电泳、琼脂糖电泳等水平：琼脂电泳、琼脂糖电泳等 垂直：垂直： 聚丙烯酰胺凝胶电泳聚丙烯酰胺凝胶电泳用用 途途 提供蛋白质分子量、电荷、亚基结构、分离纯化提供蛋白质分子量、电荷、亚基结构、分离纯化 蛋白质的纯度；蛋白质的纯度； 对蛋白复杂混合物的定性分析；对蛋白复杂混合物的定性分析； 了解不同生理病理时特定组织或细胞中蛋白质的了解不同生理病理时特定组织或细胞中蛋白质的 变化；变化； 适用范围广泛。适用范围广泛。 特特 点点 成本低：设备、人力、时间成本低：设备

4、、人力、时间 灵敏度高：可检测灵敏度高：可检测g、甚至、甚至ng水平的量水平的量 分辨率高：可分离数百、甚至上千种不同成分分辨率高：可分离数百、甚至上千种不同成分 重复性好：可重复重复性好：可重复 可信度高：罕有假象发生可信度高：罕有假象发生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)蛋白质电泳类型蛋白质电泳类型 按材质按材质 滤纸电泳、淀粉胶电泳、醋酸薄膜电泳、滤纸电泳、淀粉胶电泳、醋酸薄膜电泳、 琼脂电泳、琼脂糖电泳、琼脂电泳、琼脂糖电泳、 聚丙烯酰胺凝胶电泳、毛细管电泳聚丙烯酰胺凝胶电泳、毛细管电泳 按形状按形状 圆盘（圆盘（disc）、平板（）、平板（slab）l按电泳条件按电泳条件 恒压、恒流、常压、高压恒压、恒流、常压、高压 聚

8、丙烯酰胺凝胶电泳聚丙烯酰胺凝胶电泳polyacrylamide gel electrophoresis 变性电泳：变性电泳：sodium dodesyl sulphate-plyacrylamide gel electrophoresis (sds-page) 非变性电泳：非变性电泳：native (buffer) gels 等点聚焦：等点聚焦：isoelectric focusing (ief) gels 双向电泳：双向电泳： two dimension polyacrylamide gel electrophoresis (2d-gels) 凝胶制作凝胶制作原理原理丙烯酰胺丙烯酰胺 单体单

9、体甲叉双丙烯酰胺甲叉双丙烯酰胺 交联剂交联剂过硫酸氨过硫酸氨 加速剂加速剂四甲基乙二胺四甲基乙二胺 催化剂催化剂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 （筛孔效应）（筛孔效应）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、巯基乙醇巯基乙醇 ） 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 连续连续 非连续非连续ph 8.8 gelph 8.8 gelph 8.8 gelph 8.8 gel，分离胶分离胶ph 6.8 gel, ph 6.8 gel, 浓缩胶浓缩胶适用于提取的样品蛋白浓适用于提取的样品蛋白浓度较高，加样体积较少。度较高，加样体积较少。适用于提取样品蛋白浓度适用于提取样品蛋白浓度较低，需要加样体积较大较低，需要加样体积较大sds-pagesds-page 凝胶配制：凝胶配制： 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。 分离胶缓冲液（分离胶缓冲液（tris-cl, ph 8.8）：）： 7.2 g tris、1 g sds 溶于水，盐酸调溶于水，盐酸调ph=8.8，加水至，加水至100 ml。 浓缩胶缓冲液（浓缩胶缓冲液（tris-cl, ph 6.8）：）： 3 g tris、 0.1 g sds 溶于水，盐酸调溶于水，盐酸调ph=6.8，加水至，加水至100 ml。 电泳缓冲液：电泳缓冲液： 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（交联度）（交联度）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常见问题与解决方案常见问题与解决方案l胶不平？胶不平？l凝胶漏液？凝胶漏液？n胶板洗刷干净胶板洗刷干净n加入加入aps和和temed的量要合适。的量要合适。n加入试剂后摇匀，使其充分混加入试剂后摇匀，使其充分混 合，防止部分胶块聚合不均匀。合，防止部分胶块聚合不均匀。n温度合适，受热不均匀导致胶聚温度合适，受热不均匀导致胶聚 合不均匀。合不均匀。n两块玻璃板底部要对齐。两块玻璃板底部要对齐。sds-page常见问题与解决方案常见问题与解决方案p条带比正常的条带比正常的 窄？窄？p“微笑微笑”或或“倒倒 微

18、笑微笑”条带？条带？l凝胶聚合不均匀，灌胶时候尽凝胶聚合不均匀，灌胶时候尽 量混合均匀，动作轻缓。量混合均匀，动作轻缓。l拔梳子要迅速，清洗加样孔要拔梳子要迅速，清洗加样孔要 小心，以免把上样带扭曲。小心，以免把上样带扭曲。l样品盐浓度过高会挤压其他条样品盐浓度过高会挤压其他条 带导致宽窄不一，纯化样品，带导致宽窄不一，纯化样品， 调整盐浓度。调整盐浓度。l胶板底部有气泡会影响电泳效胶板底部有气泡会影响电泳效 果，应赶走气泡。同时注意电果，应赶走气泡。同时注意电 泳槽装置是否合适。泳槽装置是否合适。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蛋白质的染色蛋白质的染色 （1）总蛋白的染色方法总蛋白的染色方法 正染：阳离子染料正染：阳离子染料 (如考马斯亮兰，如考马斯亮兰，coomassie brilliant blue) 负染：金属阴离子负染：金属阴离子 (如咪唑锌，如咪唑锌，imidazole-zinc) 银染

22、：硝酸银银染：硝酸银 其他：荧光染色或标记，放射标记（其他：荧光染色或标记，放射标记（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(丝氨酸丝氨酸) and phosphothreonine (苏氨酸苏氨酸)(but not of phosphotyrosine) . insoluble calcium phosphate to insoluble nitrophospho-molybdate complex by treated with ammonium molybdate. cutting ja, r