Hi! My target protein does not bind to the column. Before purification, I have confirmed that it has a His-tag by Western blot. Also, the protein is in the insoluble fraction so I carried out purification under denaturing conditions. Any more suggestions? Greatly appreciate them.
Thanks!
Hello,
To favor the binding of a His-protein to the column, increase the salt concentration (800 mM NaCl, for instance) could be an option.
In addition, if the protein is insoluble it could be hiding the His-tag, so to use urea could help to expose the tag, therfore favoring its binding to the matrix.
Good luck,
Carlos.
Could you please describe all the procedure? Need to provide details for people to help you
I never performed Western blot for his tag, but confirmed by DNA sequencing if it's in frame. so I don't know if Western is that reliable, as I have read that these antibodies also gives signal with non specific proteins. Did you also confirmed the gene sequence?
Hi! Thanks for all your suggestions. So this is the procedure that I used:
1. I confirmed that my protein has a His-tag thru Western blot. I used an anti-6x His-tag antibody.
2. For cell lysis, I used 50 mM NaH2PO4, pH 8.0 + 300 mM NaCl and I solubilized the insoluble fraction using the same buffer + 8 M urea. Basically, I used a Protino Ni-TED pre-packed column and just followed their protocol.
Additional info - the His-tag of my target protein is located at the C-terminal and I saw somewhere that if the C-terminal is "inside" the protein structure then most probably the His-tag is also hidden. But I used a denaturing condition so the protein should be unfolded.
And I attached here the photo of the SDS-PAGE and PVDF membrane. I got good expression and signal.
8M Urea isn't always effective. You might try 1-2M Non-detergent sulfobetanes. Never used this on a Ni-His tag column, however, so don't know if it will work.
Can you attach and sds-page image of the chromatographic results? Please include sample added, flow through, and elution.
No histidine-tagged protein in the purified fractions
• Buffer/sample composition is incorrect: The protein is found in the FT material. Check pH and composition of sample and binding buffer. Make sure that the conc. of chelating or strong reducing agents, as well as imidazole, in the solution is not too high.
• Conc. of imidazole in the sample and/or binding buffer is too high: The protein is found in the flowthrough (FT) material. Decrease the imidazole conc.
• Elution conditions are too mild (histidine-tagged protein still
bound): Elute with an increasing imidazole gradient or decreasing pH to determine the optimal elution conditions.
• The protein has precipitated in the column: Try detergents or changed NaCl concentration or elute under denaturing (unfolding) conditions (use 4 to 8 M urea or 4 to 6 M Gua-HCl) to remove precipitated proteins. For the next experiment, decrease amount of sample, or decrease protein concentration by eluting with a linear imidazole gradient instead of imidazole steps.
• Nonspecific hydrophobic or other interaction: Add a nonionic detergent to the elution buffer (e.g., 0.2% Triton X-100) or increase the NaCl conc.
• Target protein may not be histidine-tagged as expected: Verify DNA sequence of the gene. Analyze samples taken before and after induction of expression with, for example, anti-His antibodies in Western blotting.
• Histidine-tag may be insufficiently exposed: The protein is found in the FT material. Perform purification of unfolded protein in urea or Gua-HCl as for inclusion bodies. To minimize dilution, solid urea or Gua-HCl can be added to the
sample.
Source: Ni Sepharose high performance manual
You need to check the conditions of your binding buffer as suggested above. Particularly, the effect of salt concentration and pH of your binding buffer is very critical.
The signal on your Western blot is very weak. How much did you dilute your sample compared to the SDS-PAGE of your non induced and induced samples?
As WB is much more sensitive than Coomassie-stained gel, you should see a strong signal if your protein is in high quantity, which seems to be the case according to your SDS-PAGE.
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