Originally Posted by Chitra
Hi,i fused 2 genes,cloned and expressed in BL21.1 gene of 62 KDa and other gene of 60 KDa (totally fusion protein of 122 KDa).I checked with one antibody(62 KDa) in western blot,surprisingly instead of 122KDa band size ,I got only 62 KDa band size!!! Even if i use one antibody,my blot sud come in 122 KDa is it? Say me why??
After cloning my fusion gene I digested and checked.Cloning is ok.
The protein could be getting cleaved in half in the bacteria. Proteolysis can be a real problem when expressing eukaryotic proteins in bugs. Is there some kind of linker peptide between the two genes? perhaps try modifying the linker.
I had the problem that my protein has a domain at its N-terminus and a domain at its C-terminus, but the middle is predicted to be largely unstructured. When expressed in bacteria the protein gets chopped in half into the two stable domains. That might be what happens to your protein.
If you leave your blot down for a really long time you might be able to see a faint band at 122 kDa.