Fusion tagged protein and Western Blot problem - (Apr/11/2011 )
I have a Myc tagged protein. When I first received the plasmid, transfected, etc, I was able to see the Myc tagged protein band during Western, but now when I run it, I am not able to see the band. When I blot with the antibody raised against my specific protein, I am able to pick up the band with no issue.
Is there any reason why the myc Ab won't detect my myc tagged protein?
I thought I was diluting it too much but even with a small amount, I still can't get anything.
Any comment's would be greatly appreciated!
Antibody has gone off? Do you have another protein that is also myc tagged that you can use to check whether it is the antibody or the protein?
There is a possibility that during this time I have been working with my protein, the myc tag might have broken off?
Not necessarily broken, but perhaps your plasmid is degraded by freeze/thaw or DNAse action. It could also be that the plasmid has been mutated by the bacteria while you were growing it up.