Methods Digest, Vol 19, Issue 7 to WS Organ Trypsin digestion

[Jess]

Message: 4
Date: 7 Dec 2006 14:03:47 -0800
From: "WS" 
Subject: How to inactivate proteins in organ extracts?
To: methods At net.bio.net
Message-ID: <1165529027.587932.93080 At 73g2000cwn.googlegroups.com>
Content-Type: text/plain; charset="iso-8859-1"

Dear Experts!

How to inactivate proteins in an aqueous organ extract? An attempt to
digest them with trypsin failed, probably due to trypsin inhibitor from
blood / serum present in the samples. Heating / boiling comes into my
mind, but this might kill other unknown substances in the extract.

I would like to tell if a certain effect of this extract is due to a
protein or something else.

Any ideas?

Thanks for your input!

Wolfgang



Dear WS,

First, I would like to say I am not an expert, I have done some work with liver extracts though. Trypsin can be funny. I have had some troubles too- but I have a lline on a kit that has a protocol for digests of organ extracts. ProteoExtract™ All-in-One Trypsin Digestion Kit
Cat. No. 650212, EMD biosciences. This protocol does involve heating though and a number of harsh reagents (TCA).

Also, a paper that might help you is INTRACELLULAR DIGESTION: The Enzymes and Anti-Enzymes Concerned Opie Physiol. Rev..1922; 2: 552-585. This talks about differences in organs  (in terms of proteins that can inhibit trypsin etc). 

Maybe a simpler way is just to filter/dialyze the proteins out of your sample? You can do this at RT (or 4 degrees), and stay aqueous, that wouldn't hurt your sample. I'm a fan of mechanical removal whenever possible for organ fractionation... Ultracentrifugation, filtration, etc, but mostly because I am after proteins that are temp sensitive and fussy if I mess with them too much chemically. Wish you success!

Cheers,
Jess White
University of Texas- Austin
Dept of Medicinal Chemistry
Hook 'em Horns!


 
---------------------------------
Everyone is raving about the all-new Yahoo! Mail beta.