making antibody

buchberg at CALVIN.JCI.TJU.EDU buchberg at CALVIN.JCI.TJU.EDU
Sat Oct 16 12:08:35 EST 1993

Dear bionetters

I am polling for your favorite protocol for making antibodies in bunnies from a prokaryotic fusion protein.  Basically, we made a fusion using a MBP vector. OUr protein of interest is ~ 25 K.  We have the fusion protein purified (i.e. isolated from a maltose column, ~2 mg) and am wondering how next best to proceed.  When the protein is blotted, we see our protein and several smaller proteins (all reacting to maltose antibody). ANd also one larger band on coomassie staining  (i.e. protein isn't completely pure). 
Several questions are:
1) Should we cleave off the MBP part and purify our protein of interest
2) How much protein product is needed.
3) Should the protein be further purified on a preparative acrylamide gel, if so  HOW best to do it?
4) Any other advice or suggestions are welcome.        

Thanks in advance for all responses 

Art Buchberg

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