How many His residues? - (Sep/21/2007 )
I'm currently purifying a 6x His tagged protein and like many others, I'm getting a lot of contaminants from the cell culture in my sample. So I'd like to increase the affinity of my tag to the nickel column by increasing the number of His residues in the tag, so I can use more stringent wash conditions. However, I'm not sure how many residues would be ideal. I was thinking 10, but is there a point of diminishing return? And are there any new problems associated with using large stretches of His residues? Thanks.
-jimmunology
-jimmunology-
QUOTE (jimmunology @ Sep 21 2007, 06:10 AM)
I'm currently purifying a 6x His tagged protein and like many others, I'm getting a lot of contaminants from the cell culture in my sample. So I'd like to increase the affinity of my tag to the nickel column by increasing the number of His residues in the tag, so I can use more stringent wash conditions. However, I'm not sure how many residues would be ideal. I was thinking 10, but is there a point of diminishing return? And are there any new problems associated with using large stretches of His residues? Thanks.
-jimmunology
-jimmunology
Your problem would be a protein degradation, just add some protease inhibitor!
Adding few Histidine residues also costs your time, you can use other expression system such as GST fusion.
-NTH-