I have a problem in western blot that I can't resolve by myself. When I am use to add 100 microgram of proteins for each sample but after running and transfer its impossible to me to see bands of protein. What can be the reason of that? Thank you for your help
From what you say, I can only give a few advices:
- For the quantification, make sure your sample does not contain any substances which disturb the assay. The Bradford assay is for example sensitive against SDS or DTT. See here for more details. This prevents you from loading a sample without enough protein.
- Make sure that you really have protein on your gel. Run a gel as normal and then stain it with Coomassie Blue (it cannot be used for western afterwards).
- Don't loose your sample - this happens easier than most people think.
- Make sure your transfer works. This can either be done by using pre-stained ladders (which are helpful anyway) or by doing Ponceau Red staining. This can be washed off with water and doesn't disturb downstream applications.
- Depending on the type of membrane you are using, they might need pre-treatment. PVDF membranes are extremely hydrophobic and need to be activated in methanol.