Hi Jack,
I wonder that one should not be doing the assay before trying out the
system.
One assumes that all the components of the Ab stages are OK.
You appear to be working nearly blind, so go get DAKOs method book in pdf
format if you need the basics, dakocytomation.co.uk is the place this side
of the water.  dakocytomation.us over there
 
Trypsinisation, for twenty odd years we have been using 40mg of Trypsin in
100ml of 0.1% Calcium Chloride (0.04%)8 to 12 mins digestion,   So your
0.25% seems more than strong enough.
I wonder at the EDTA, which would appear to chelate any Calcium that might
be around as a co-enzyme.


Dave
Manchester UK  

-----Original Message-----
From: Jack England [mailto:joeamateur@hotmail.com]
Sent: 19 February 2004 19:06
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] while we're talking about von Willebrand Factor...


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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