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HIS-tag Protein Problems
6X His-tag on a 148kDa large capsid protein
Here's the rough protocol:
Sonication in Lysis Buffer : 50mM HEPES pH 7.5, 1% Tween-20, 10% glycerol, 250mM NaCl, 10mM 2-mercaptoethanol - Roche EDTA free protease inhibitor added
Sonicate pellet in Wash Buffer: 20mM Tris-HCl, 250mM NaCl, 2M Urea, 1% Tween-20 pH 8.0
Pellet Solubilization Buffer: 20mM Tris-HCl, 250mM NaCl, 5mM Imidazole, 6M GuHCl, 1mM 2-Mercaptoethanol pH 8.0 for at least 24 hours at room temp
Most of the protein comes out in the nickel column Flow Through (analyzed via SDS-PAGE) and when doing a western blot with Penta His barely anything is detectable, in comparison to the corresponding SDS-PAGE. What does elute is weird. I get some off with 25mM Imidazole, then some more with 40mM then some more with 250mM. I don't bump up the gradient until the first concentration has done 10CV and I actually get 3 peaks.
So here's the survey and would love to get opinions on what the hell is happening to the HIS tag.
1.I do not use Protease inhibitors in the 2M Urea wash step or solubilization. Could the HIS tag be getting attacked? I ran the EXPASY peptide cutter and there are locations but I thought just in the initial lysis was enough but maybe following denaturation I am subjecting it to more?
2.Protonation of the HIS? - PI of the protein is 6, pH of the buffer is 8, it's a 6X His so that seems off.
3. Hydrophobic? Following the 6M GuHCl could the HIS tag actually be occluded in a hydrophobic poket?
4. Aggregation of the protein even following 6M GuHCl?
Any other thought or are these my best options to follow?
Re: HIS-tag Protein Problems
Before all of that you mentioned:
Check your linker in the construction. Be sure that you add a large linker!
|histag , problems , protein|
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