Biotinylation problems-need advice.

Reno Pontarollo pontreno at duke.usask.ca
Sat Jan 21 11:17:49 EST 1995


Greetings,

	I have recently puified the bovine serum protein hemopexin.  I 
have biotinylated it twice useing the Clonetech and Boehringer kits.  The 
Mr of the labelled protein is 10 kDa greater than the unlabelled 
protein.  However, I cannot get streptavidin::AP or streptavidin::HRP to 
bind the labelled protein.  The other possibility is that binding does 
occur, but the hemopexin::biotin/streptavidin::AP-HRP complex somehow 
impairs the function of the AP or HRP.  The streptavidin::AP-HRP is 
active when it binds transferrin::biotin.
	Has anybody else had this problem?  Do you have an 
explanation/solution?
	I am now trying to directly conjugate hemopexin to AP and HRP as 
an alternative method.  I would appreciate any insites you may have?

P.S.  Has anybody used that radio-labelling kit from Amersham that uses 
[35-S] instead of [125-I]?  How does it work?

Thanks, Reno



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