my protein is fussed with GST tag (mol. wt ~ 52 kDa). i am using during Ni-NTA (binding in presence of 10 mM Imidazole and elution with 500 mM imidazole ). I am seeing non specific bands after washing with 30 CV with buffer.
further, after cleaving GST tag and again passing through column; flow through still shows non specific protein binding to my protein of interest.
I also tried step gradient but issue still persist.
in attachment: gel pic: lane 1: Ni-NTA purifired fraction; lane 2; protein after GST tag cleaved: lane 3: Flow through after passing cleaved protein through nickel column
Are you running a slow, linear imidazole gradient for desorption? Are you using a reducing agent during purification? Is you protein prone to oligomerization?
@omair Gonzalez Ortega
No sir, I am not running linear imidazole concentration.
I gave binding and washing in 10mM imidazole and elution with 500 mM.
Further I cleaved GST tag and again passed to Ni NTA column.
Further, this protein does not form oligomers.
I have tried reducing and non reducing condition, but no change.
IMAC is a pseudo-affinity technique that, as you proved, has reduced selectivity. You need to increase the initial imidazole concentration and run a slow imidazole gradient to improve separation. See if this helps.
A slow linear gradient of imidazole for elution will help provide a protein that is more pure, but it may not solve the problem. You may resort to using very small amount of non-ionic detergents in the buffer to remove those proteins that may be binding non-specifically to your protein. This will help elute those proteins before you elute the desired protein.
Thank you @ Gustavo.
using linear gradient of imidazole didn't solved the issue. However, I performed HIC which lead to 95 % pure protein of interest.
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