western blotting - (Jul/06/2004 )

Hi:

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.

Thanks

Somebody asked the same question some time ago, check this topic

http://www.protocol-online.org/forums/inde...?showtopic=2889