HELP! Protein purification in presence of gelatin/BSA

tina guina tguina at WESLEYAN.EDU
Fri Oct 4 12:00:35 EST 1996


Dear colleagues!
I am trying to purify a small cationic and hydrophobic protein from 
bacterial culture supernatants. The culture medium contains insect a 
mammalian cell culture components including 50g/L of BSA and 0.4-0.5% 
GELATIN.
The protein of interest is very likely to be produced in small amounts 
and associating with one or more medium components of higher MWt.
When expressed in E. coli, this protein is soluble, but it partitions 
into the detergent phase after the triton X114 extraction.
Biologically, this protein is a cytolysin which inserts into the target 
membranes and form pores.

I need to show the presence of this protein in culture supernantants. I 
have a good affinity purified antiserum against the protein.
My questions are:
1) what would be the best method to concentarte the protein?
	  (is ammonium sulfate good enough and would it precipitate the gelatin 		    
    as well)
2) is there a simple, for the protein harmless way to get rid of gelatin 
without heating the solution?

Thanks a lot in advance!

Tina Guina



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