western blotting of expressed protein - (Jul/02/2004 )
I have inserted my DNA fragments (500kb and another is 1500kb) into an AAV-IRES-hrGFP vector. This vector contains DNA sequences encoding for green fluorescent protein which will be used as an indicator of expression of my inserted fragment. After transfection with these two kinds of plasmid, I want to find out if these plasmids can be expressed in cells (293T).
Green fluorescent can be seen easily in the cultured cells. This usually means that my fragment is expressed together with GFP separately. (In the same mRNA but the proteins are separated with each other)
Then I test the cell lysate by western blotting. But I cannot find one of the two proteins I want.
I don¡¦t know the reason.
The structure of the plasmids is correct. The rate of the cultured cells which express GFP is nearly 30%. And I get good results in positive and negative control in western blotting.
hi, ixsix: where did you buy the vector AAV-IRES-hrGFP from and what does the AAV mean? I currently using the clontech/BD biosciences vector pIRES and I cloned EGFP and another protein of interest cDNA into MCSa and MCSb site of the pIRES plasmids, but there is always a problem to express the EGFP from the MCSB site. Did you have this kind of problem? By the way, the hrGFP was already in the vector when you bought it, or you cloned the hrGFP into the MCSB site of the AAV-IRES vector? Thanks!