Hi
to check if the protein exist in the cells and the medium ( cell culture and transfection), we conduct western blot . at the start we saw the protein yet when we repeat the experiment with the same material we didn't see the protein .
the sample from the transfection we stored at -80 C.
we don't know what is the problem!
Did you add a protease inhibitor? How did you Lyse the cells? Would you expect the protein to cytosolic/soluble or membrane bound? How much did you load on the gel?
You can repeat the western blot with a fresh sample. If you get results, it means there is a problem with the storage conditions. There is a possibility that the protein can degrade over time, so add protein stabilizers before storing the sample. The protein can also aggregate if it has a propensity to do so. Mild detergents can be used to prevent this. Additionally, avoid freeze-thaw cycles more than once. If you need to use the sample multiple times, aliquot the sample before freezing.
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