Factor Xa digest

Dr. David Martin david.martin at read.my.sig
Wed Jan 29 07:57:18 EST 1997


In article <E4rpqB.KKt at fsa.bris.ac.uk>,
   bimvh at zeus.bris.ac.uk (MV. Hayes) wrote:

+>
+>I am trying to improve my Factor Xa digest of my fusion protein and 
+>would be greatful if anyone has any useful hints and/or conditions.
+>
+>At present I am digesting with 1% Factor Xa in 50Mm sodium acetate 
ph8 
+>(the digest is done in Sodium acetate to avoid dialysis after the 
digestion 
+>prior to loading on to a Mono S column) for 6hrs at 37C.
+>

A few hints. Ensure there is no detergent in the mix. FXa is rather 
intolerant of detergent.
Drop the pH by a little (to about 7-7.4).
Add a little calcium (2 mM)
Add a little salt (to 150mM or so)

You can dilute out the salt before loading onto the MonoS column.

You can check the activity of your FXa with a synthetic substrate. 1% 
sounds like a lot of FXa.

Hope this helps.

.d

* David Martin - Atherosclerosis and Thrombosis research group  *
* http://www.uio.no/~damartin/      david.martin at biotek.uio.no  *
* Lab +47 22 95 84 54 Fax +47 22 69 41 30  GSM +47 90 74 27 65  *



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