Protein Immunoblotting- An Introduction  to Western Blotting
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Protein Immunoblotting- An Introduction to Western Blotting

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Western blotting is an important technique used in cell and molecular biology. The specificity of the antibody-antigen interaction enables a target protein to be identified from a complex protein ...

Western blotting is an important technique used in cell and molecular biology. The specificity of the antibody-antigen interaction enables a target protein to be identified from a complex protein mixture. Western blotting can produce qualitative and semi-quantitative data about that protein. This powerpoint will attempt to explain the technique and theory behind western blot.

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Protein Immunoblotting- An Introduction  to Western Blotting Protein Immunoblotting- An Introduction to Western Blotting Presentation Transcript

  • 1) Eelectrophoretic separation of protein or of nucleic acid fragments in the sample 2) Transfer to and immobilization on a matrix. 3) The probe is added to the matrix to bind to the target molecules. 4) Any unbound probes are then removed. 5) Visualization of bound probe
  • Western blot Protein Detection
  • • Detects proteins and estimates their molecular weight. • Detects changes in phosphorylation and lipid modifications. • Used to detect changes in protein expression.
  • Sample Preparation SDS-PAGE (Protein Electrophoresis) Electro-transferring Immunoblotting
  • • Major components of the sample loading “buffer” – SDS – DTT – Tracking dye – Glycerol – Protease inhibitor
  • Western Blot Visual Protocol: Phase 1: Sample Preparation
  • • Sodium dodecyl sulfate (SDS) • Tris buffer (either glycine or tricine) • Acrylamide and NN-bis-acrylamide – Forms gel matrix • TEMED – Catalyst for polymerization (produces free radials from APS) • Ammonium persulfate (APS) – Source of free radials for polymerization Could purchase pre-cast gels if you have the money. Ingredients in Gel
  • Stacking (concentrating) gel 4% acrylamide 0.5M Tris-H+Cl-, pH 6.8, 0.1% SDS Resolving (separating) gel 10% acrylamide (36.5:1, acryl/bis) 1.5 M Tris-H+Cl-, pH 8.8, 0.1% SDS Running buffer 0.25 M Tris base; 1.92 M glycine, pH 8.3; 1% SDS Resolving gel Stacking gel Discontinuous system
  • How to Make an SDS-PAGE gel
  • Western Blot Visual Protocol: Phase 2: Protein Electrophoresis
  • • uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane • Transfer buffer contains: Tris, Glycine, and methanol but no ions. • Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings • Western blot transfer can be done in wet or semi- dry conditions
  • • Equilibrate gel in transfer buffer in separate tray. • Equilibrate filters and sponges in transfer buffer. • PVDF membranes must be soaked in methanol, before equilibration in transfer buffer. Nitrocellulose membranes are soaked directly in transfer buffer  Mount transfer sandwich in blotting chamber which already contains transfer buffer
  • Western Blot Visual Protocol: Phase 3: Membrane Transfer
  • • Washing (TBS-T) • Blocking • Incubation with Primary antibody • Washing (TBS-T) • Incubation with secondary antibody
  • • Blocking reduces nonspesific binding of antibody (primary or secondary) to protein or membrane • Too little => high background • Too much reduces the signal • Incubation time: – 1-2 hrs at RT with shaking • Blocking agents – Fat free dry milk – Bovin serum albumin – Casein – Gelatin – Hemoglobin – Ovalbumin • Buffer: – PBS, phosphate buffered saline, pH 7.5-8.0 – TBS, TRIS-buffered saline, pH 7.5
  • • After blocking membrane, add antibodies in to blocking solution (ie 5% milk). • Incubate overnight at 4oC or 2 hours at room temperature.
  • • Buffer: PBS w/Tween 20 or TBS w/Tween 20 – TW20 concentration must be determined for each antibody and antigen – Usually 0.01-0.2% • Time: Number of washes and duration of each wash must be determined in each case – Usually 3X5 min – Use large buffer volume: 50-100 ml for 8X10 cm membrane – Incubation with vigorous shaking
  • • Buffer: same as for primary antibody • Dilution must be determined in each case – Usually 1:1,000 - 1:100,000 • Incubation time must be determined in each case – Varies from 5 min to 2 hrs enzym
  • • Colorimetric detection • Chemiluminescent detection • Radioactive detection • Fluorescent detection
  • Western Blot Visual Protocol: Phase 4: Immunoblotting