Hi Lisa,
Have you compared your yield of purified protein of soluble protein per gram of pellet, 4M Guanidine solubilized pellet (wash and elute from Ni column with 8M urea .5M imidazole..otherwise the gel with run crappy), and and total protein in the pellet on a page gel? The main reason I would think you get high background of cellular proteins on you Ni purification is because your yield is too low (I know obvious..). There are a variety of methods to increase yield like expressing at RT (assuming your expressing in E.coli), or switching to yeast, insect, or mamallian cells if your protein is of human origin. The most helpful and easiest method to try first (after trying low temp), would be switching the tag from N to C terminus or vice versa. Otherwise switch to a thermophile version of your protein if possible (and try cysteine mutants or other bacterial organisms for source the
source gene).
Paul
ps one thing I forgot, you mention it is a DNA binding protein, solublizing in 2M NaCl is much better than 1M NaCl, you could just be losing it...the protein that is :-)
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From: LISA <[log in to unmask]>
To: [log in to unmask]
Sent: Thursday, August 4, 2011 11:51 AM
Subject: [ccp4bb] gst-tag protein purify problem
hi guys,
I have a DNA binding protein and expressed the DNA binding domain (150 aa) with his-sumo tag or gst tag at the n-terminal. I tried to purified it with Ni column or Gst column separately. But purity is lower than 50% after Ni or GST column. This protein only stable with 1M Nacl or higher. I worked on it almost half year. But I still can not get the pure protein.
please give me some suggestions. Thank you.
Lisa