双向电泳的样品制备

1. 双向电泳的样品制备 有效避免样品损失

2. -大鼠舌组织(26 µg) • -pH 3-10, 13cm • -无样品处理. • 在一向等电聚焦过程中Bromophenol dye没有发生移动. • Contributed byJanice Cheung,GE-Healthcare Malaysia

3. -大鼠舌组织(26 µg) -pH 3-10, 13cm -样品经过 Ettan™ 2DClean Up Kit处理. -SDS-PAGE胶最后用 Plus One Silver Staining kit染色 Contributed byJanice Cheung,GE-Healthcare Malaysia

4. 样品的准备步骤 • Cell disruption • Protein precipitation • Solubilization • Protection against protease activities • Removal of • nucleic acids • lipids • salts, buffers, ionic small molecules • insoluble material

5. 在样品制备前需要考虑的问题 • 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 representation

6. 细胞破碎的方法 • Freeze-thaw or osmotic lysis • Detergent lysis • Sonication • Enzymatic lysis • French pressure cell • Grinding (mortar and pestle) • Mechanical homogenization

7. 现在常用在样品预分离的方法 • By solubility • sequential extraction with increasingly strong solubilizing agents (???) • By chromatography • e.g. hydrophobic interaction (???) • By centrifugal separation of subcellular components • e.g. mitochondria, nuclei, cytosol • By affinity • Immunoprecipitation of complexes or selective removal of abundant proteins

8. 干扰物质 • Proteases • Nucleic acids • Polysaccharides • Plant phenols • Lipids • Salt ions • Insoluble material

9. 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 buffer

10. 常用的蛋白酶抑制剂 • PMSF inhibits serin- and thiol-proteases • - is not stable in aqueous solutions • - is inactivated by DTT, ß-mercaptoethanol etc. • - is toxic (alternative: Pefablock™) • AEBSF (Pefabloc™)alters pI of some proteins • EDTA inhibits metallo-proteases • Pepstatin inhibits acidic proteases • high pH inhibits/slows down actions of proteases • Limits: Protease inhibitors do not inactivated all proteases! Proteins may become modified by protease inhibitors

11. 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 in silver stained 2-D patterns • Removal 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 ultracentrifugation • Removal of polysaccharides: • TCA/acetone precipitation of proteins and re-solubilization • ultracentrifugation

12. 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 urea • DNase I will show up on a 2-D map. (pI ~5, MW ~30 kDa) • Benzonase (both DNase and RNase activity) is also commonly used. • Sonication works very well!

13. Effect of DNase Treatment E. coli extract on 7 cm pH 3-10 NL + DNase - DNase

14. 质脂 • May interact with membrane proteins, “consume” detergents and form insoluble precipitates • Remedies: • 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. 盐离子 • High concentrations of salt ions interfere with IEF and cause overheating, “empty lanes”, disturbed 2-D gel patterns • Desalting: • 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) • “electrophoretic 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 hours • Limits: possible loss of proteins

16. Effect of salt E. coli extract pH 4-7 no salt 30 mM NaCl

17. Dialysis Spin dialysis Gel filtration Precipitation/resuspension Slow Detergents can concentrate with protein Protein losses Complicated, can cause losses De-salting techniques

18. Effect of dialysis Pre-dialysis sample Dialyzed sample pH 5 6 7.5 10 pH 5 6 7.5 10

19. Desalting by Low Voltage IEF Bovine vitreous proteins • 150 V / 30 min 100 V / 5 hrs

20. Chaotropes • Urea • - efficiently breaks hydrogen bonds • - typically used as 8 to 9.5 M solution • Thiourea • - weakly soluble 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 • proteins

21. 7 M urea / 2 M thiourea Extraction:Comparison Urea vs Urea/Thiourea 8 M urea Rat liver

22. Inhibits proteolysis Useful with lipid-rich samples Limited to low sample load Can disturb first dimension SDS in 2-D Sample Prep Best solubilizing agent known but not compatible with IEF unless diluted into an excess of another detergent

23. CHAPS vs. Amidosulfobetaine-C12 Spinach thylakoid membranes 2% CHAPS* 2% ASB C-12* kDa 212 170 116 76 pH 7 10 7 10 *Extraction/rehydration also contained 7 M urea, 2 M thiourea, 0.5% Pharmalyte 3-10, 60 mM DTT

24. DTT (dithiothreitol) DTE (dithioerythreitol) 2-mercaptoethanol Tributylphosphine(TBP) Triscarboxyethylphosphine (TCEP) triscyanoethylphosphine most commonly used interchangeable with DTT required at high concentration, contains impurities, but may have solubilization benefits (?). Poorly soluble, very hazardous Good reductant, but negative charge makes it unsuitable for 1st dimension. Uncharged, soluble, but efficacy as reductant is in doubt. Reductants

25. Sample treatment is very important • Cleanup from contaminants • Dissolve complexes completely • Protein-protein • Protein-polysaccharides • Stop protein activities (protease, phosphatases) • Precipitation is the most efficient…..

26. Protein precipitation • Clean-up from lipids, nucleic acids, polysaccharides, polyphenols, salts • Concentration of proteins • Irreversible inhibition of proteases • Prevention and dissolution of complexes • For DIGE: removal of endogeneous peptides

27. Ammonium sulfate Not efficient, de-salting necessary, (salting out); NOT recommended TCA precipitation Can be hard to re-solubilize Acetone and/or ethanol Leaves SDS behind, but many proteins not precipitated TCA plus acetone(Damerval et al. 1986) More effective than either alone, good for basic proteins Chloroform and methanol(Wessels and Flügge, 1984) Time consuming, largevolumes necessary Cleanup Kit (TCA, detergent, acetone) 1 ½ hours, very efficient, good recovery Protein precipitation procedures

28. E. coli lysate precipitated with TCA/acetone and resuspended Crude E. coli lysate Effect of sample precipitation

29. Protein solubilization • ideal procedure: • disruption of all non-covalently and S=S-bound protein complexes and aggregates into a solution • ideal buffer: • cleaves all S=S-bridges, ionic bonds, H-bonds and hydrophobic interactions under conditions compatible with IEF and without modifying proteins

30. 蛋白质的有效溶解常用试剂 • Urea (8-9.8 M) , or 7 M urea / 2 M thiourea • Detergent (CHAPS,…) • Reductant (DTT, 2-mercaptoethanol) • Carrier ampholytes (0.8 % IPG buffer) • Sonication can help solubilization • Sample can be heated only prior to addition of urea • Full solubilization may require several hours.

31. 促进样品复溶的有效方法 • Pellet must not become completely dry! • Pipette repeatedly lysis solution over the pellet (do not vortex!) • Rehydration can take several hours (or over night) @ RT (do not vortex!) • Carefully sonicate (avoid heating of sample) • Use PlusOne Molecular grinding kit • Freeze pellet with lysis solution at 20 °C • Use SDS solution (2 % SDS, hot) and then dilute with 9 M urea / 4 % CHAPS

32. 细菌 酵母(或其他真菌类) 培养的细胞 植物样品 High nucleic acid/protein ratio. Nucleic acid removal techniques are often employed Tough 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 be 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. 特殊类型的样品

33. Effect of sample prep technique(Drosophila larva extract) Homogenate precipitated with 80% acetone, 10% TCA. Resuspended in 8 M urea, 4% CHAPS Homogenized in 8 M urea, 4% CHAPS Homogenized in 2% SDS Heated at 95 ºC 3 min First dimension is pH 3-10 L run on IPGphor in 8 M urea, 2% CHAPS, 0.5% IPG buffer, 65 mM DTT

34. New Sample Preparation Kits from APBiotech • PlusOne Molecular Grinding Kit • PlusOne 2-D Clean-up Kit • PlusOne 2-D Quant Kit • PlusOne Microdialysis Kit • PlusOne PAGE Clean-up Kit (not for 2-D electrophoresis)

35. 2 D Clean-Up Kit for first-dimension IEF • Acidic precipitation with detergent co-precipitant • Washing of pellets withaddition of organic solvents • Resuspension of pellets insample solution

36. Rat liver extracted with 4 % SDS, 40 mM Tris base Untreated Treated with PlusOne 2-D Clean-Up Kit pH 4-7 pH 4-7 2 D Clean-Up Kit for first-dimension IEF

37. Untreated Treated with PlusOne 2-D Clean-Up Kit Cell extract Stasyk T, Hellman U, Souchelnytskyi S. Optimizing sample preparation for 2-D electrophoresis. Life Science News 9 (2001) 9-12.

38. Compatible with... PlusOne 2 D Quant Kit

39. PlusOne Microdialysis Kit • 3 h to overnight

40. Effect of dialysis Pre-dialysis sample Dialyzed sample pH 5 6 7.5 10 pH 5 6 7.5 10

41. E.coli extract undialyzed dialyzed with PlusOne Mini Dialysis Kit pH 3-10 NL pH 3-10 NL Effect of dialysis

42. DeStreak Reagent & DeStreak Rehydration Solution

43. DeStreak 20 µg reduced mouse liver proteins applied anodic to Immobiline DryStrips pH 6–11, 11cm. Reswelling solution containing either DTT (A & B) or DeStreak (C & D). A & C:10.6 kVh; B & D: 25.3 kVh.

44. Comparison of two 2-D electrophoresis maps generated with and without DeStreak in the strip rehydration buffer.

45. Conclusions • Reproducibility is enhanced ; • Reproducible spot pattern is generated with strips covering different pH ranges; • A more cathodic position is observed for the majority of spots; • the spot pattern is completely stable over time in the first dimension; • The 2nd dimension is unaffected enabling the application of conventional protein identification/characterization techniques.