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Effect of Triton on IMAC - Low affinity?

Effect of Triton on IMAC - Low affinity? - Protein Forum

Effect of Triton on IMAC - Low affinity? - Protein Forum


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Old 09-20-2010, 03:41 AM
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Default Effect of Triton on IMAC - Low affinity?



Working on an insoluble 6X HIS Tagged HSV protein via a denaturation protocol.
Briefly:
Following lysis - Wash of insoluble pellet in 2M Urea/500mM NaCl/1% Tween-20/20mM Tris, pH 8
Followed by extraction in : 8M Urea, 20mM Tris, 500mM NaCl, 1mM b-ME, 5mM Imidazole, 1% Triton X-100, pH 8
Extraction is applied to nickel IMAC resin at a flow rate of 50cm/hr to 75cm/hr
IMAC Equilibration buffer is : 6M Urea, 20mM Tris, 500mM NaCl, 1mM b-ME, 5mM Imidazole, pH 8
and I am seeing about 20% the protein of interest within the flow through.
What I have done:
Optimized the NaCl concentration to 250-500mM NaCl in Extraction and IMAC equilibration buffers. I was using 2M NaCl in the equilibration at one point to reduce non-specific column interactions but that resulted in a lot of on-column protein aggregation, so 500mM NaCl works well.

So my big question is...I know GE IMAC resin can handle 2% Triton on its own but I believe that it is protein specific. So has anyone witnessed having a high (1% in this case) concentration of Triton X-100 in the protein load sample result in a lowered affinity towards IMAC?

I have also demonstrated that 1% triton X-100 in the extraction is not really necessary and I could easily go lower in concentration.

I am planning this experiment now in the absence and presence of Triton X, but if someone could provide some insight it would be greatly appreciated.
I have read in most papers that 0.1% to 0.05% are commonly utilized.

Oh, another thing I am seeing is that my protein begins to elute at 20mM imidazole! Related?

Thank you
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