We purified a protein with yellowish brown color From Ni-NTA Column, but I don't know why. The protein with 70 amino acid contains 12 cysteines. And I want to seek the protocol that how to denature and renature the Metallothionein? Thanks For your HELP.
Some of the Ni probably became reduced during the chromatography, causing it to elute from the NTA resin. You should be able to remove it by dialysis or gel filtration or ultrafiltration, or by running the purified protein fraction over an uncharged NTA column (after first removing any reducing agent).
@Adam B Shapiro, Hi, buddy. I am benefited from your wonderful answer. I have ultrafiltrated to remove the Ni-ions and salt. But it does not change. As the increasing of protein concentration, the color seems become dark than before. I am confused the disulfate bond and metal ions and how to resolute the problem? AND I know Proteins that contain iron-sulfu is yellow.
If you still have the brown-colored contaminant in your sample despite dialysis, first try to remove it by centrifugation. If that doesn't work, try either ion exchange chromatography or gel filtration chromatography to remove it.
Are both gels loaded with the same amount of protein? Overloading can lead to broad bands.
Did your gel sample buffer have a reducing agent to reduce any disulfide bonds in the protein? Failure to do so could lead to anomalous migration.
Hi,
Did you solve the problem? The brown color of the solution may appear due to the presence of DNA. After lysis lots of DNA in the lysate give this color. You could try ion exchange/RP HPLC. Ammonium sulfate precipitation method also helps to remove the DNA.
If you are worried about protein degradation, use protease inhibitor to save your target protein from being chopped by enzymes.
Remove the color by centrifugation or by ion exchange chromatography
@ Moaed E. Algazally and Abhigyan Sengupta, thank you all, buddy. YOUR suggestion is very helpful for me. I will try the protocol that you mentioned. BUT I'm confused the rich disulfate peptide. Why some of them could binding with ions, but some don't. And the color of protein I purified seem become light, gradually.
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