Can blotted enzymes retain there activity on nitrocellulose?

marko fennema at chem.rug.nl
Tue Dec 17 08:15:41 EST 1996


Hello Newsreaders,

I want to detect a complex of streptavidin coupled via a biotin moiety to
an other protein. In principle I could do this via a western blot using 
a non denaturating gel and antibodies against the steptavidin. But I think 
that there must be faster ways to do this. I have been brainstorming about
this and thought of some experiments on which I would like to have some 
comments.

1- Using a fluorecent labeled streptavidin (fluorescein-strep.), can this 
be seen under UV in the acrylamide gel? or only with a fluorecense microscoop?

2- Using a streptavidin-alkaline phosphatase complex which is commercially
available, and incubating the gel with a colouring substrate. Is it possible 
to do the staining in a gel?

3- Blotting the same steptavidin-AP to a nitrocellulose mebrane, and incubate 
the NC-membrane with the colouring substate. Do the electrostatic interactions 
between the blotted enzyme and the membrane inhibit enzymatic catalysis?


thanking you in advance for your comments,

Marko Fennema
 



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