biotinylated 2nd Ab+Streptavidin/HRP?

Agustin Hernandez ahernan at cica.es
Wed Jul 18 04:16:12 EST 2001


I am trying to detect a membrane protein in western blots, but, since it
is present in such ridiculous amounts in the cell, I have not been able to
do it so far. Also, because we could only tag it with a 6xHis tag,
immunoprecipitation is not very satisfactory (our antiHis antibodies are
just fair). I know the protein is there, because we can purify it (in
almost negligible amounts I must say, but functional and recognisable by
the antiHis Ab). Membrane fractionation did not help in detecting it.
I have been told that western blots based on biotinylated secondary
Ab+Streptavidin-HRP could be a solution to see this protein on western.
The guy who told me this didn't actually know much about it (and neither
do I!). Does anybody have a clear idea on how Normal secondary Ab-HRP
westerns compare to the biotin/streptavidin-HRP system? Does anyone have a
good protocol I could use? 



ahernan at cica.es
Inst. Rec. Nat. Agrobiol. (CSIC) Seville, Spain

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