The best way to optimize Western blots is to run 2 gels that are the same sample loaded in all lanes.
-Then you can transfer these to membrane.
- cut these membranes into 4s (be careful to cut between the lanes). mark the tops (this is required to know where the size markers are).
- optimize each cut piece differently but keep everything else the same change 1 thing at a time
- block with BSA for half, block with Milk 5% for the other half
- vary 10 primary antibody
- vary secondary a bit
- if signal is weak incubate with 10 in 4C overnight
More information on optimizing your Western Blot
hope this helps
Molecular station admin