I have been working on optimizing western blotting for Caspr (contactin associated protein), but I am having difficulties and would like suggestions.
- I ran my proteins on a 40% acrylamide gel,
- I used TBST for washing and BSA as blocking buffer.
- I incubated the blot with primary antibody overnight.
- The secondary antibody is linked with HRP.
I could not find any bands in the blot.(Same problem was faced in case of contactin) What might be the reason?
I repeated the experiment a couple of times and found that secondary antibody(Anti-mouse,1:5000)wasn't working anymore.
I made freshly diluted secondary antibody again and found bands.
The old secondary antibody dilution was only few months old.What may have gone wrong with it?