Western Blot
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Target group: MD Biochemistry and Microbiology Students, Students of Life sciences, Biotechnology, and Biochemistry Slides prepared on Nov 2016
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Western Blot
- 1. Western Blotting ASHIKH SEETHY SR and PhD Scholar Dept of Biochemistry AIIMS- New Delhi PG PRACTICAL
- 2. Overview 2 ▸3 Elements 1. Separation of proteins 2. Transfer to a solid support 3. Marking target protein using a proper primary and secondary antibody to visualize
- 3. Overview 3 ▸Southern Blot ▸Northern Blot ▸Western Blot ▸Eastern Blot ▸South-Western Blot ▸Dot Blot
- 4. Overview 4 ▸Protein Extraction ▸Protein Quantification ▸Sample Preparation ▸Electrophoresis ▸Blotting ▸Blocking ▸Staining ▸Development of the Blot ▸Data Analysis ▸Applications
- 6. Cell/Tissue lysis 6 Considerations: ▸To collect all the proteins of interest ▸Enrichment can be done using sub-cellular fractionation ▸Denaturing vs Non-denaturing ▸Lysis Buffer ▹pH ▹Salt ▹Detergents ▸Mechanical interventions if required ▸Done in cold temperature ▸Protease and phosphatase inhibitors
- 7. Some Common Detergents: 7 ▸NP-40: Nonidet P-40 ▸Triton-X-100 ▸SDS ▸Sodium deoxycholate ▸Ethyl-trimethyl ammonium bromide
- 8. RIPA Buffer 8 ▸Radio-Immuno-Precipitation Assay lysis buffer ▸Components and Functions: 150 mM sodium chloride 1.0% NP-40 or Triton X-100 0.5% sodium deoxycholate 0.1% SDS (sodium dodecyl sulphate) 50 mM Tris, pH 8.0 Which buffer will you use for co-immuno-precipitation?
- 9. Lysis Buffers: 9 ▸What more will you add to the lysis buffer? Protease and Phosphatase inhibitors
- 10. Protease Inhibitors : 10 ▸Serine proteases Phenyl methyl sulfonyl fluoride (PMSF) Aprotinin Leupeptin ▸Cysteine proteases Leupeptin ▸Aspartic proteases Pepstatin ▸Metallo-proteases EDTA EGTA
- 11. Phosphatase Inhibitors : 11 ▸Serine/Threonine phosphatases Sodium Fluoride Sodium pyrophosphate ▸Tyrosine Phosphatases Sodium orthovanadate When will you abstain from adding Phosphatase Inhibitors?
- 13. Protein Quantification: Method Wavelength Sensitivity Principle Spectrophotometry 260 and 280 nm 210 nm 1-10 μg/L Aromatic amino-acids and peptide bonds absorb particular wavelengths Lowry 660 nm 10 μg/L Cu(2+) binding to proteins to from to Cu(+) which reacts with Folin reagent Bradford 595 nm 20 μg/L Dye-binding method BCA 562 nm 0.5 μg/L Cu(2+) binding to proteins to from to Cu(+) which reacts with BCA 13 Which is the most accurate method?
- 14. Protein Quantification: 14 30-40 μg of protein is loaded for electrophoresis/well y = 0.0011x - 0.0085 R² = 0.9989 y = 0.0011x - 0.0085 R² = 0.9989 y = 0.0011x - 0.0085 R² = 0.9989 y = 0.0011x - 0.0085 R² = 0.9989 Absorbance Concentration in µg/mL BCA after Compatable Protein Preparation
- 16. Laemmli 2X Buffer 16 ▸4% SDS ▸10% 2-mercapto-ethanol ▸20% glycerol ▸0.004% bromophenol blue ▸0.125M Tris HCl ▸pH: 6.8 95-100 °C for 5 minutes or 70 °C for 5-10 minutes
- 18. SDS-PAGE 18 ▸Acrylamide ▸N,N-methylene bisacrylamide ▸SDS ▸APS ▸TEMED ▸Buffer Pore size is determined by: ▸Total amount of acrylamide present (T) Varies inversely with acrylamide concentration ▸Percentage of N,N-methylene bisacrylamide (C) Minimum pore size at C= 5% of T !!Acrylamide is a potent cumulative neurotoxin
- 19. Disc Electrophoresis 19 ▸Isotachophoresis ▸What all are discontinuous? 1. Gel structure 2. pH of the buffer 3. Nature of ions in the gel and the electrode buffer ▸ Samples are loaded ▸ Mol. Weight markers
- 20. Other Types of Protein Electrophoresis: 21 ▸2D electrophoresis ▸Native PAGE ▸Non-reducing PAGE ▸Gradient PAGE
- 22. Membranes: 23 Nitrocellulose ▸Hydrophobic interaction ▸Brittle Poly-vinylidene-di-fluoride (PVDF) ▸Strong hydrophobic interaction ▸Reprobing ▸Requires “activation” in methanol Pore size: ▸0.1, 0.2 or 0.45µm
- 23. Transfer: 24 ▸Diffusion blotting ▸Vacuum blotting ▸Tank (Wet) electrotransfer ▸Semi-dry electrotransfer
- 24. Towbin’s Transfer Buffer: 25 Glycine Tris Base Methanol Water SDS-Optional Discontinuous buffer systems
- 25. Ponceau S Stain: 26 ▸Reversible ▸Limited sensitivity ▸Does not photograph well ▸Fades quickly ▸Documentation difficult ▸Stain the gel
- 26. Blocking 27
- 27. Blocking: 28 Why? ▸ To prevent any nonspecific binding of antibodies to the surface of the membrane How? ▸Bovine serum albumin ▸Non-fat milk ▸Casein ▸Serum ▸Non-protein blocking reagents In TBS/PBS with or without detergent Which one to use?
- 28. Staining 29
- 29. Choice of Antibody: 30 ▸Choose the epitope ▸Sample species ▸Host species ▸Label ▸Monoclonal vs Polyclonal ▸Direct vs Indirect ▸Dilutions ▸Primary antibody ↓ ▸Wash ↓ ▸Secondary antibody ↓ ▸Wash
- 31. Detection Systems and Labels: 32 ▸Colorimetric ▸Chemi-luminescence ▸Fluorescence ▸HRP ▸ALP
- 32. Colorimetric Systems- HRP 33 •Decreased sensitivity compared to AP •Fading of blots upon exposure to light •Inhibition of HRP activity by azide •Nonspecific color precipitation
- 36. Data Analysis 37
- 37. Western Blot is Semi-quantitative: 38 ▸Loading and transfer rate can vary ▸Loading control: GAPDH, β-actin, Tubulin, PCNA Choosing control: ▸Sub-cellular localisation ▸Molecular weight ▸Nature of the experiment How to stain? ▸Simultaneous staining with target antibody ▸Cutting the blot ▸Stripping the blot
- 38. Applications 39
- 39. Further Reading: 40 ▸Western Blotting - A Beginner’s Guide from Abcam ▸Protein Biology Learning Center- Thermoscienctific ▸Bio-rad- Introduction to Western Blotting ▸Voet and Voet- 4th ed ▸Wilson and Walker- 7th ed
- 40. Thank you 41
Editor's Notes
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid