I am doing whole purification in 6M GuHCl, but I am not able to elute the protein with 500mM Imidazole. Is there any precipitation of Imidazole in presence of GuHCl ?
Imidazole cannot be used to elute the target protein in denaturing purification using 6M GuHCl, pH gradients are used instead. You can elute your target protein using 6M GuHCl buffer pH4.5. For example most commonly used denaturing sample
buffer is 100 mM NaH2PO4, 10 mM Tris–HCl, 6 M GuHCl, pH 8.0; denature washing
buffer is 100 mM NaH2PO4, 10 mM Tris–HCl, 6 M GuHCl, pH 6.3 and denature elution
buffer is 100 mM NaH2PO4, 10 mM Tris–HCl, 6 M GuHCl, pH 4.5.
Dear Paras
Guanidinium hydrochloride at 6 M is protein-denaturing. Precipitation of your protein of interest in its presence can be checked before smple application onto the affinity column you use.
Q1: What is it your objective? Purification of the protein in a denatured form?
Q2: What is it the pH value you use for the chromatography
Dear Joseph Kreit,
I want to purify protein in denaturing conditions, and after that check on western blot.
I am not using columns, I am doing it in eppendorfs.
I am doing chromatography
Dear Paras
The proposed buffers (Huanting Liu answer) to elute your protein in a denatured form seem to be right for your experiment. Try using them. Thanks to keep us informed.
Q1: How do you separate liquid from the resin in eppandorf? By centrifugation?
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