雙向電泳的樣品制備GE公司

1、雙向電泳的樣品制備雙向電泳的樣品制備有效避免樣品損失2 /GE Title or job number /4/14/2022-大鼠舌組織 (26 g) -pH 3-10, 13cm-無(wú)樣品處理.-在一向等電聚焦過(guò)程中Bromophenol dye沒(méi)有發(fā)生移動(dòng).-Contributed byJanice Cheung,GE-Healthcare Malaysia3 /GE Title or job number /4/14/2022-大鼠舌組織 (26 g) -pH 3-10, 13cm-樣品經(jīng)過(guò) Ettan 2DClean Up Kit處理.-SDS-PAGE膠最后用 Plus One Sil

2、ver Staining kit染色Contributed byJanice Cheung,GE-Healthcare Malaysia4 /GE Title or job number /4/14/2022樣品的準(zhǔn)備步驟樣品的準(zhǔn)備步驟 Cell disruptionProtein precipitationSolubilizationProtection against protease activitiesRemoval of nucleic acids lipids salts, buffers, ionic small molecules insoluble material5 /GE

3、 Title or job number /4/14/2022在樣品制備前需要考慮的問(wèn)題Is the sample from cells or solid tissue?Is pre-fractionation desired?What kind of interfering substances are present?Quality of separation vs. total protein representation6 /GE Title or job number /4/14/2022細(xì)胞破碎的方法Freeze-thaw or osmotic lysis Detergent ly

4、sisSonication Enzymatic lysisFrench pressure cellGrinding (mortar and pestle)Mechanical homogenization7 /GE Title or job number /4/14/2022現(xiàn)在常用在樣品預(yù)分離的方法By solubility sequential extraction with increasingly strong solubilizing agents (?)By chromatography e.g. hydrophobic interaction (?)By centrifugal

5、separation of subcellular components e.g. mitochondria, nuclei, cytosolBy affinity Immunoprecipitation of complexes or selective removal of abundant proteins8 /GE Title or job number /4/14/2022干擾物質(zhì)干擾物質(zhì) Proteases Nucleic acids Polysaccharides Plant phenols Lipids Salt ions Insoluble material9 /GE Tit

6、le or job number /4/14/2022如何使蛋白酶失活如何使蛋白酶失活Problem: Cell lysis endogenous proteases set free proteolytic attack artifacts (Mr, pI)Remedies: protease inhibitors (2-D Protease Inhibitor Mix) precipitation reaction (2-D Clean Up Kit) boiling with SDS buffer10 /GE Title or job number /4/14/2022常用的蛋白酶抑制劑

7、 PMSFinhibits serin- and thiol-proteases- is not stable in aqueous solutions- is inactivated by DTT, -mercaptoethanol etc.- is toxic (alternative: Pefablock) AEBSF (Pefabloc)alters pI of some proteins EDTAinhibits metallo-proteases Pepstatininhibits acidic proteases high pHinhibits/slows down action

8、s of proteasesLimits:Protease inhibitors do not inactivated all proteases!Proteins may become modified by protease inhibitors11 /GE Title or job number /4/14/2022核酸和多糖核酸和多糖 cause horizontal and vertical streaks in 2-D patterns increase sample viscosity clog pores of PAGE may cause background smear i

9、n silver stained 2-D patternsRemoval of nucleic acids: treatment with a protease-free DNAse/RNAse mixture TCA/acetone precipitation of proteins and re-solubilization adding a basic polyamine (e.g. spermine) and ultracentrifugationRemoval of polysaccharides: TCA/acetone precipitation of proteins and

10、re-solubilization ultracentrifugation12 /GE Title or job number /4/14/2022除去核酸的方法DNase I and RNase A are commonly used(add 0.1x vol of 1 mg/ml DNase I, 0.25 mg/ml RNase A in 50 mM MgCl2)Nucleases will not work in 8 M ureaDNase I will show up on a 2-D map. (pI 5, MW 30 kDa)Benzonase (both DNase and R

11、Nase activity) is also commonly used.Sonication works very well!13 /GE Title or job number /4/14/2022Effect of DNase TreatmentE. coli extract on 7 cm pH 3-10 NL+ DNase- DNase14 /GE Title or job number /4/14/2022質(zhì)脂質(zhì)脂May interact with membrane proteins, “consume” detergents and form insoluble precipit

12、atesRemedies: organic solvents (ethanol) phenol protein precipitation (e.g. TCA/acetone, 2-D Clean Up Kit)Limits: loss of proteins (some may not re-solubilize)15 /GE Title or job number /4/14/2022鹽離子鹽離子High concentrations of salt ions interfere with IEF and cause overheating, “empty lanes”, disturbe

13、d 2-D gel patternsDesalting: minidialysis, spin dialysis or gel filtration (proteins can get lost) protein precipitation (Ettan 2D Cleanup Kit, TCA-acetone); resolubilization with lysis buffer “in-gel desalting”: dilute the sample and apply a larger volume instead (rehydration loading preferably) “e

14、lectrophoretic desalting I”: remove salt ions by appropriate electrode papers in a gel-side up focusing device (Ettan IPGphor Manifold, Multiphor II) soft sample entry: low voltage for several hoursLimits: possible loss of proteins16 /GE Title or job number /4/14/2022Effect of saltE. coli extract pH

15、 4-7no salt30 mM NaCl17 /GE Title or job number /4/14/2022De-salting techniquesDialysisSpin dialysisGel filtrationPrecipitation/resuspensionSlowDetergents can concentrate with proteinProtein lossesComplicated, can cause losses18 /GE Title or job number /4/14/2022Effect of dialysisPre-dialysis sample

16、Dialyzed samplepH566107.557.510pH19 /GE Title or job number /4/14/2022Desalting by Low Voltage IEF150 V / 30 min 100 V / 5 hrsBovine vitreous proteins20 /GE Title or job number /4/14/2022ChaotropesUrea- efficiently breaks hydrogen bonds- typically used as 8 to 9.5 M solutionThiourea- weakly soluble

17、in water (1 M), but in concentrated urea solutions (2 - 2.5 M)- typically 2 M thiourea and 5 - 8 M urea- superior solubilizing power, especially for nuclear and membrane proteins21 /GE Title or job number /4/14/2022Extraction:Comparison Urea vs Urea/Thiourea7 M urea / 2 M thioureaRat liver8 M urea22

18、 /GE Title or job number /4/14/2022SDS in 2-D Sample PrepInhibits proteolysisUseful with lipid-rich samplesBest solubilizing agent known but not compatible with IEF unless diluted into an excess of another detergentLimited to low sample loadCan disturb first dimension23 /GE Title or job number /4/14

19、/2022CHAPS vs. Amidosulfobetaine-C12kDaSpinach thylakoid membranespH2% CHAPS*2% ASB C-12*Extraction/rehydration also contained 7 M urea, 2 M thiourea, 0.5% Pharmalyte 3-10, 60 mM DTT7107102121701167624 /GE Title or job number /4/14/2022ReductantsDTT (dithiothreitol)DTE (dithioerythreitol)2-mercaptoe

20、thanol Tributylphosphine(TBP)Triscarboxyethylphosphine(TCEP)triscyanoethylphosphinemost commonly usedinterchangeable with DTTrequired at high concentration, contains impurities, but may have solubilization benefits (?).Poorly soluble, very hazardousGood reductant, but negative charge makes it unsuit

21、able for 1st dimension.Uncharged, soluble, but efficacy as reductant is in doubt.25 /GE Title or job number /4/14/2022Sample treatment is very important Cleanup from contaminantsDissolve complexes completelyProtein-proteinProtein-polysaccharidesStop protein activities (protease, phosphatases)Precipi

22、tation is the most efficient.26 /GE Title or job number /4/14/2022Protein precipitationClean-up from lipids, nucleic acids, polysaccharides, polyphenols, saltsConcentration of proteinsIrreversible inhibition of proteasesPrevention and dissolution of complexesFor DIGE: removal of endogeneous peptides

23、27 /GE Title or job number /4/14/2022Protein precipitation proceduresAmmonium sulfate Not efficient, de-salting necessary,(salting out); NOT recommendedTCA precipitationCan be hard to re-solubilizeAcetone and/or ethanolLeaves SDS behind, but many proteins notprecipitatedTCA plus acetone(Damerval et

24、al. 1986)More effective than either alone, good forbasic proteinsChloroform and methanol(Wessels and Flgge, 1984)Time consuming, large volumes necessaryCleanup Kit (TCA, detergent, acetone)1 hours, very efficient, good recovery28 /GE Title or job number /4/14/2022Effect of sample precipitationCrude

25、E. coli lysateE. coli lysate precipitated with TCA/acetone and resuspended29 /GE Title or job number /4/14/2022Protein solubilizationideal procedure:disruption of all non-covalently and S=S-bound protein complexes and aggregates into a solutionideal buffer:cleaves all S=S-bridges, ionic bonds, H-bon

26、ds and hydrophobic interactions under conditions compatible with IEF and without modifying proteins30 /GE Title or job number /4/14/2022蛋白質(zhì)的有效溶解常用試劑蛋白質(zhì)的有效溶解常用試劑 Urea (8-9.8 M) , or 7 M urea / 2 M thiourea Detergent (CHAPS,) Reductant (DTT, 2-mercaptoethanol) Carrier ampholytes (0.8 % IPG buffer)Soni

27、cation can help solubilizationSample can be heated only prior to addition of ureaFull solubilization may require several hours.31 /GE Title or job number /4/14/2022促進(jìn)樣品復(fù)溶的有效方法Pellet must not become completely dry!Pipette repeatedly lysis solution over the pellet (do not vortex!)Rehydration can take

28、several hours (or over night) RT (do not vortex!)Carefully sonicate (avoid heating of sample)Use PlusOne Molecular grinding kitFreeze pellet with lysis solution at 20 CUse SDS solution (2 % SDS, hot) and then dilute with 9 M urea / 4 % CHAPS32 /GE Title or job number /4/14/2022特殊類型的樣品細(xì)菌酵母(或其他真菌類)培養(yǎng)的

29、細(xì)胞植物樣品High nucleic acid/protein ratio. Nucleic acid removal techniques are often employedTough cell walls require vigorous disruption techniques. Protease activity is high. SDS is usually used.Salt carry-over from growth medium or wash solution can be significant. Salt-free buffer/osmoticum should b

30、e used for washing (10 mM Tris / 250 mM sorbitol pH 7.0).Dilute source of protein. Precipitation is usually employed. Protease activity is high. Reductants and inhibitors are used to prevent phenolic modification.33 /GE Title or job number /4/14/2022Effect of sample prep technique (Drosophila larva

31、extract) Homogenized in 8 M urea, 4% CHAPSFirst dimension is pH 3-10 L run on IPGphor in 8 M urea, 2% CHAPS, 0.5% IPG buffer, 65 mM DTTHomogenized in 2% SDSHeated at 95 C 3 minHomogenate precipitated with 80% acetone, 10% TCA. Resuspended in 8 M urea, 4% CHAPS34 /GE Title or job number /4/14/2022New

32、 Sample Preparation Kits from APBiotechPlusOne Molecular Grinding KitPlusOne 2-D Clean-up KitPlusOne 2-D Quant KitPlusOne Microdialysis KitPlusOne PAGE Clean-up Kit (not for 2-D electrophoresis)35 /GE Title or job number /4/14/20222 D Clean-Up Kit for first-dimension IEFAcidic precipitation with det

33、ergent co-precipitantWashing of pellets withaddition of organic solventsResuspension of pellets insample solution36 /GE Title or job number /4/14/20222 D Clean-Up Kit for first-dimension IEFRat liver extracted with 4 % SDS, 40 mM Tris baseUntreatedTreated with PlusOne 2-D Clean-Up KitpH 4-7pH 4-737 /GE Title or job number /4/14/2022Cell extractUntreatedTreated with PlusOne 2-D Clean-Up KitStasyk T, Hellman U, Souchelnytskyi S. Optimizing sample preparation for 2-D electrophoresis.Life Science News 9 (2001) 9-12.38 /GE Title or job number /4/14/2022PlusOne 2 D Quant KitCompatible with