expression of fusion protein - how to lysis membrane protein (Apr/29/2005 )

hi, everyone!

Recently, I constructed a recombinant plasmid which could express

fusion protein (X-EGFP) after being transfected into mammalian cells.

However, when 48h after the transfection the cells were lysed, and

Western blot was performed, surprisingly, the molecular weight of the

interest band was not correct, that is to say, it demonstrate the MW of

EGFP. By the way, my target protein is a membrane protein.

I want to know why I can only obtain the reporter protein instead of the

fusion protein, and the method of cell lysis is wrong? If you know what it

happens, please share with me. Thank you very much!

What is the molecualr weigth of your fusion protein? What might have happened is that your protein construct had the EGF infront of the N-terminus and thus it is being cleaved as your protein is targeted to the membrane. To check this try solublize your membrnaes with 1% SDS at room temperature for 5 minutes and then run on on an SDS-PAGE and test for its presences by Wb. I hope this helps, good luck!

Dear bassamfahmawi,

Thank you for your kindly reply! And I will have a try according to your

suggestions.

By the way, the molecular weight of my fusion protein is about 50 kd.