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.
Text of Protein Immunoblotting- An Introduction to Western Blotting
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.
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