I want to compile a good Western Blot Background Troubleshooting guide.
Does any one have experience in this area? So far I have several problems of Western Blotting Background with solutions: 1) Too much secondary antibody used causing high background
Solution - use less secondary antibody (try 1:10,000 or more)
2) Wash time during washes (between antibodies etc) too short
Solution - increase your washing time
3) Wrong western blot membrane type used
Solution - use the right membrane type. For minimum background, try using PVDF membranes for western blot transfer.
4) Blocking Reagent (non-fat dry milk) is too diluted
Solution - Make sure you properly prepare FRESH nonfat dry milk (5% w/v) dissolved in TBST or suitable buffer such as PBS.
5) Contamination or Dirty membrane and/or blotting equipment
Solution - A) Use clean equipment, freshly prepared buffers, and new membranes.
B) Always avoid handling and touching membranes with bare
hands (even with gloves handle very little). Make sure you use gloves and forceps to handle western blot PVDF membranes.
please post your inputs here