Blot, Blot, Western Baby Kristin B. Dupre June 30th, 2011

Western Blotting “Blotting” = transfer of biological samples from a gel to a membrane and subsequent detection on surface of membrane Routine technique for protein analysis Introduced by Towbin et al. (1979) Also called immunoblotting

Pre-Western Blotting Tissue preparation Bradford assay Homogenize tissue in lysis buffer and draw off supernatant Bradford assay Total amount of protein in each sample is determined Normalization of sample Each sample needs to have a proportionate concentration of total protein

Step 1: Gel Electrophoresis Separation of proteins by isoelectric point, molecular weight, electric charge, or a combination of these factors Most common type is SDS-PAGE Sample proteins are covered in negatively charged SDS and move to positively charged electrode through acrylamide mesh of gel

Step 2: Electro-Transfer Transfer proteins to membrane Make proteins accessible to antibody detection Electric current pulls proteins from gel into PVDF or nitrocellulose membrane

Step 3: Blocking Block non-specific binding Typically use 2-5% bovine serum albumin (BSA) or non-fat dry milk

Step 4: Detection with Antibodies Primary Antibody Binds to antigen Wash to remove unbound primary Secondary Antibody Binds to primary antibody and reacts with substrate

Step 5: Chemiluminescent Detection Substrate reacts with HRP-conjugated secondary antibody Luminescence is produced in proportion to amount of protein present on membrane Expose membrane to x-ray film

you get beautiful data like Karen! And if you’re lucky… you get beautiful data like Karen!

Step 6 (optional): Stripping Wash to remove chemiluminescent substrate Incubate in Restore Stripping Buffer Wash to remove buffer Repeat steps 3-5 with different primary

The End