蛋白质－临床博士.ppt

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1、蛋白质研究的技术与策略,任惠民,自报家门（任惠民）,毕业：华东师范大学 化学系原工作单位：中国科学院上海生理研究所97年引进至（原）上海医科大学神经病学研究所国外工作：日本杏林大学，美国加州大学承担基金：国家、省部、中科院、重点实验室等发表论著：180余篇,研究蛋白质的意义,蛋白质的重要性 构成生命体的最重要物质 与生命活动紧密相关 蛋白质与疾病的关系 表达水平与功能改变导致疾病发生,内 容,蛋白质研究的前处理技术蛋白质的分离技术蛋白质的凝胶电泳技术Western blot（蛋白免疫印迹）蛋白质组学Proteomics,蛋白质的凝胶电泳技术,电泳（Electrophoresis）,电泳是指带电

2、粒子在电场中向与自身带相反电荷的电极移动的现象。（1）类型 液相：在液相介质中 固相：固相中或固相表面（2）凝胶电泳 水平：琼脂电泳、琼脂糖电泳等 垂直：聚丙烯酰胺凝胶电泳,用 途,提供蛋白质分子量、电荷、亚基结构、分离纯化 蛋白质的纯度；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 变化；适用范围广泛。,特 点,成本低：设备、人力、时间灵敏度高：可检测g、甚至ng水平的量分辨率高：可分离数百、甚至上千种不同成分重复性好：可重复可信度高：罕有假象发生,Structure and Properties of Protein,PROTEINS-polymer of ami

3、no acids with biological activity made of alpha amino acid(20)STRUCTURE of Amino Acids aas have a carboxyl group(-COOH)&amino group(-NH2)and are often ionized at physiological pH,Effect of pH and buffer on protein charge,Proteins are amphoteric compounds and are therefore either positively or negati

4、vely charged Depending on the pH of their local enviroment Post translation modificationsthe addition of charged and uncharged sugarthe addition of phosphate groupsulphydryl cross-link Isoelectric Point-pH where there is no net charge in molecule,Migration of protein depends on,Strength of electric

5、field(Heat)net charge on molecule(pH)size and shape of molecules(choice of support)Properties of supporting medium(viscosity,electroendosmosis),蛋白质电泳类型,按材质 滤纸电泳、淀粉胶电泳、醋酸薄膜电泳、琼脂电泳、琼脂糖电泳、聚丙烯酰胺凝胶电泳、毛细管电泳按形状 圆盘（disc）、平板（slab）按电泳条件 恒压、恒流、常压、高压,聚丙烯酰胺凝胶电泳polyacrylamide gel electrophoresis,变性电泳：Sodium dodes

6、yl sulphate-plyacrylamide gel electrophoresis(SDS-PAGE)非变性电泳：Native(buffer)gels等点聚焦：Isoelectric focusing(IEF)gels双向电泳：Two dimension polyacrylamide gel electrophoresis(2D-gels),凝胶制作原理,丙烯酰胺 单体,甲叉双丙烯酰胺 交联剂,过硫酸氨 加速剂,四甲基乙二胺 催化剂,Native gel electrophoresis,For the detection of particular proteins(i.e.,an e

7、nzyme)on the basis of its biological activityPolyacrylamide(normally 7.5%gel),but the SDS is absent and the proteins are not denatured prior loadingProteins separate according to their different electrophoretic mobilities and the sieving effect（筛孔效应）of the gelThe enzyme of interest can be identified

8、 by incubating the gel in an appropriates substrate,SDS-PAGE(SDS-Polyacrylamide gel electrophoresis),Separate protein according to sizeSamples must be previously boiled 5 minutes in sample buffer containing:SDS(CH3-(CH2)10-CH2OSO3-Na+),1 molecule binds every 2 amino acids residues-Mercaptoethanol（-巯

9、基乙醇）Sucrose or GlycerolIonizable 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 HCl,SDS-PAGE,The original charge on protein is masked by the negatively charged of SDS,SDS-PAGE,连续 非连续,pH 8.8 gel,pH 8.8 ge

10、l，分离胶,pH 6.8 gel,浓缩胶,适用于提取的样品蛋白浓度较高，加样体积较少。,适用于提取样品蛋白浓度较低，需要加样体积较大,SDS-PAGE,凝胶配制：30 g 丙烯酰胺、1 g Bis-丙烯酰、1 g SDS 溶于水至100 ml。分离胶缓冲液（Tris-Cl,pH 8.8）：7.2 g Tris、1 g SDS 溶于水，盐酸调pH=8.8，加水至100 ml。浓缩胶缓冲液（Tris-Cl,pH 6.8）：3 g Tris、0.1 g SDS 溶于水，盐酸调pH=6.8，加水至100 ml。电泳缓冲液：28.8 g甘氨酸、6 g Tris、1 g SDS 溶于水至1000 ml。,

11、Recommended acrylamide concentration for protein electrophoresis,Separation 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）,SDS-PAGE and CBB R-250 staining,Gradient gel

12、s,The acrylamide concentration is varied uniformly from,typically 5%at the top of the gel to 25%acrylamide at the bottom of the gel,AdvantagesGreater range of protein Mw values can be separated than on a fixed-percentage gelThe proteins with a very similar Mw values may be resolved,SDS-PAGE常见问题与解决方案

13、,胶不平？凝胶漏液？,胶板洗刷干净加入APS和TEMED的量要合适。加入试剂后摇匀，使其充分混 合，防止部分胶块聚合不均匀。温度合适，受热不均匀导致胶聚 合不均匀。两块玻璃板底部要对齐。,SDS-PAGE常见问题与解决方案,条带比正常的 窄？“微笑”或“倒 微笑”条带？,凝胶聚合不均匀，灌胶时候尽 量混合均匀，动作轻缓。拔梳子要迅速，清洗加样孔要 小心，以免把上样带扭曲。样品盐浓度过高会挤压其他条 带导致宽窄不一，纯化样品，调整盐浓度。胶板底部有气泡会影响电泳效 果，应赶走气泡。同时注意电 泳槽装置是否合适。,IFE(Isoelectric Focusing Electrophoresis),

14、separates proteins by isoelectric pointslarge pore size of gel and equilibrium conditions minimize molecular sievingnative or denaturing conditions possiblegenerates pH gradient in electric fieldgradient range depends on ampholyte pKa values anode:dilute acid(H3PO4)cathode:dilute alkali(NaOH),IFE,2D

15、-PAGE(Two-dimension polyacrylamide gel electrophoresis),Combines of IEF(separating according to charge,pI)with size separation of SDS-PAGE,2D-PAGE,蛋白质的染色,（1）总蛋白的染色方法 正染：阳离子染料(如考马斯亮兰，Coomassie brilliant blue)负染：金属阴离子(如咪唑锌，imidazole-zinc)银染：硝酸银 其他：荧光染色或标记，放射标记（fluorescent staining or labeling,and radi

16、olabeling）（2）特殊染色方法 糖基化蛋白 磷酸化蛋白,Selected staining methods,“Classical”Coomassie Brilliant Blue R-250 Stain,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:40%(v/v)ethanol and 10%(v/v)acetic acid.Coomassie R-250:reddish tint Coomassie G

17、-250:greenish tint detection limit 10100 ng Fazekas de St Groth,et al.Biochim.Biophys.Acta 1963,71:377391.,“Classical”Coomassie Brilliant Blue R-250 Staining Protocol,1.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 g

18、el briefly(12 min)with CBB R-250 destaining solution(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-25

19、0 destaining solution with shaking until 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,CBB G-250 staining solution:0.12%(w/v)CBB G-250 dye,10%ammonium sulfate,10%phosphoric acid

20、,and 20%methanol.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.Electrophoresis.1988,9:255 262.Candiano G,et al.Electrophoresis.2004,25:1327 1333.,Colloidal C

21、oomassie Brilliant Blue G-250 Staining Protocol,1.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 the gel in colloidal CBB G-250 staining solution for at least 3-4 h.Staining can continue overnight if more con

22、venient.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 incubation in CBB G-250 destaining solution,Imidazole-Zinc Reverse Stain,Imidazole solution:0.2 M imidazole containing 0.1%SD

23、S.dissolve 6.81 g imidazole and 500 mg sodium dodecyl sulfate(SDS)in 500 mL water.Zinc sulfate solution: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 detect

24、ed in SDS-PAGE gelsReverse-stained proteins can be efficiently eluted and used in biological and enzymatic assays.Fernandez-Patron C,et al.Biotechniques.1992 12:564 573.Castellanos-Serra L and Hardy E.Nat.Protoc.2006,1:1544 1551,Imidazole-Zinc Reverse Staining Protocol,1.Briefly rinse the gel(2 30 s

25、)in freshly prepared Milli Q water2.Incubate the gel in 500 mL 0.2 M imidazole 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

26、 background will become white and protein spots transparent.Note:prolonged 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 stain

27、ed gel in water at 4C until use,Silver Nitrate Stain,Fixing solution:40%(w/v)ethanol and 10%(w/v)acetic acidSensitizer:0.02%sodium thiosulfate-pentahydrate(The solution must be prepared the day of use)Silver nitrate solution:0.2%silver nitrate and 0.02%formaldehyde(37%).Developer:3%sodium carbonate,

28、0.05%formaldehyde(37%),and 0.0005%sodium thiosulfate pentahydrate.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 Stain,Today,more than 100 different variants of silver-staining protocols exist for proteins separated in polya

29、crylamide 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 sodium hydroxide).Patterns are then developed in dilute acidic solutions of formaldehyde.Acid

30、ic 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 in 40%ethanol and 10%acetic acid at least for 3 h.2.Wash the gel in 30%ethanol for 20 min,followed

31、 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 manually shaking.5.Add silver nitrate solution and gently agitate for 30 min.Protect the tray from bright light.6.Rins

32、e the gel in water for 3 20 s while manually shaking.,Fast Silver Nitrate Staining Protocol,7.Quickly add developing solution(3%sodium carbonate,37%formaldehyde).Sometimes,a gray or brown precipitate may form.Develop until protein spots are clearly visible(515 min).The most intense spots will appear

33、 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 deionized 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

34、 10 min)with Milli Q water.,Fluorescence-Based Protein Detection Methods for Total Protein Staining,Two major approaches(1)covalent derivatization of proteins with fluorophores prior to IEF(2)postelectrophoretic protein staining by the intercalation of fluorophores into the SDS micelles coating the

35、proteins by the direct electrostatic interaction with the proteins.For pre-electrophoretic fluorescent These dyes are commercially available as CyDyes,labeling are the cyanine-based dyes that react with lysyl or cysteinyl residues.For post-electrophoretic fluorescent These dyes are SYPRO Ruby and De

36、ep Purple stain,SYPRO Ruby Total Protein Stain,Fixing solution:40%(v/v)ethanol and 10%(v/v)acetic acid.SYPRO Ruby staining 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 fo

37、r multiplexed staining,combination with fluorescence detection of glyco-or phosphoproteins,SYPRO Ruby Staining 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 SY

38、PRO Ruby staining solution incubating for at least 3 h at room temperature or overnight.protect the 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

39、imager.Use the filter sets that match the excitation(maximum,450 nm)and emission(maximum,610 nm)wavelength for SYPRO Ruby.Images may also be obtained by using a simple UV transilluminator.,For Labeling Protein Samples for DIGE,SDS sample buffer:1%(w/v)SDS,100 mM Tris-HCl(pH 8.5).Thiourea/urea/CHAPS

40、buffer A:2 M thiourea,7 M urea,4%CHAPS,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 a

41、re stable at 20C for 6 monthsStop solution:10 mM lysine.,Minimal(Lys)Labeling Protein Samples,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 co

42、ncentration 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 DMF

43、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 Samples,5.Incubate the sample in the d

44、ark 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.Add 1 L of DTT solution and 1 L of Pharmalyte 3 10 per 50 L of sample solution.labeled samples can be stored for at least 3 months at 70C.9.Run IEF and SDS-PAGE.Protect from

45、 light to minimize photobleaching of the fluorescence dyes.,Pro-Q Diamond Phosphoprotein Stain,Fixing solution:40%(v/v)ethanol and 10%(v/v)acetic acid.Pro-Q Diamond phosphoprotein stain:Ready-to-use solution(Molecular Probes,Eugene,OR,USA).Pro-Q Diamond phosphoprotein destaining solution:25%acetonit

46、rile 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%ethanol and 10%acetic acid(or 50%methanol and 10%acetic acid)overnight.Replaceme

47、nt 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 min per wash).4.Incubate the gel with Pro-Q Diamond phosphoprotein stain for 23 h protect the r

48、eagent 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.Continue with image acquisition.Pro-Q Diamond stain has excitation/emission maxima of 555/580 nm A visible-light transillumin

49、ator,or(with reduced sensitivity)a 300 nm transilluminator,Phosphoproteins,Phosphorylation is a reversible widely used by cells for activation/inhibition of specific pathways both in basic energy metabolism and in specialized signal transduction processes.Phosphorylation results in a shift of the pr

50、otein 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 Methods,Methyl Green(basic dye)An old procedure depends on the hydrolysis of the phosphoester linkage of phosphose