[Histonet] while we're talking about von Willebrand Factor...

[Jack England]

Another REALLY basic question from the Rookie...

We're about to do our first vWf assay today, and none of us in this lab have 
done Ab staining before. We've got primary Ab and secondary Ab, and I'm 
reasonably certain I can get it to work once I get past the Ag retrieval 
step. For that, the only enzyme we have in the lab right now is 0.25% 
trypsin/0.1mM EDTA mix. I've tried to check the references supplied 
previously, but all require subscriptions that I don't have access to.

Here's my question: is that concentration of trypsin adequate for Ag 
retrieval, and if so, how long would it incubate for (assume 37 degrees)? 
(For that matter, would the chelative effects of the added EDTA mess things 
up?)

>From what I gather, proteinase K digestion would be the best way to go, but 
unfortunately time is of the essence and we can't get it fast enough.

So the more important question, I guess, is whether I tell my PI that it 
can't be done with what we have on hand, or whether it can be done (though 
not perfectly). Can anyone out there advise?

--Many, many thanks,
Jack England

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