Overexpression of Viral Protein in mammalian cell line - (Aug/31/2006 )
I am trying to express a His-tagged viral protein in a mammalian cell line. I am producing a His tagged protein of the correct size (confrimed by Western blot) but there is not enough protein produced to be seen on a Coomassie stained gel. I need to increase the amount of protein produced because I need to mass spec it. I have no antibody to the specific protein. Does anyone know what I could do?
I'm guessing it's not a soluble secreted protein but could you over-express on a big scale in mammalian cells then purify it on a column using the His tag. Chances are you need to do this anyway to get something pure enough to use for the mass spec. Isn't silver staining a more sensitive method for detecting proteins rather than coomasie staining?
Yes the protein is secreted into the media. I have silver stained and I think that I can see the protein band which is giving me the signal on the Western blot. However, I still have the problem that I need to be able to produce alot more protein for it to be useful to us.
try adenoviral expression system or baculoviral expression system.