Another option is to purify in denatured condition, then refold. --Chun
-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Meg
Sent: Tuesday, September 23, 2008 12:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Troubleshooting protein purification
Another suggestion that comes to mind is crosslinking via Cys, are you
purifying under reducing conditions ? Or since you have inclusion
bodies they're in general not correctly folded hence expose
hydrophobic regions and can interact with other proteins, to avoid
unspecific interaction add
Meg,
I might add that if you have boiled your samples, you might disregard
this:
1) Instead of contamination, you might just be seeing multiple
bands due to 'aggregation' of your protein! Make sure you boil the
sample prior to loading on gel and also that your loading dye
contains SDS,
Hello Meg,
Since your protein is quite acidic, your next step could be e.g. anion
exchange - provided that you are able to get the protein into a suitable
buffer w/o losing it (since you will pass through the pI). If you can, the
simplest way to do so is to add TRIS to the pH4.5 buffer until you g
Hi,
Many things can lead to your observation. Please outline all steps of
your purification procedure as it is not clear what is done before
and after the Ion Exchange steps.
I am not sure if IEF in your emails refers to Isoelectric focusing,
as the acronym is usually used??
Couple of s
