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
