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

2. 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

3. 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

4. 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

5. 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

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

7. 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

8. 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

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

10. 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

11. The End