I purified a protein containing His tag via Ni-NTA chromatography wherein the protein was eluted in elution buffer containing 500mM imidazole, 50 mM Tris, 500mM Nacl. Since my next step of purification was Ion exchange chromatography, I dialyzed the protein in dialysis buffer containing 50mM Tris and 5mM beta mercapthoethanol. However, in the third round of dialysis, I observed extreme precipitation of the protein.
Kindly let me know what can be done to reduce the precipitation.
An important question is whether the precipitation was reversible or irreversible. Irreversible precipitation is the result of denaturation of the protein, resulting in aggregation followed by precipitation. Exposure of the protein to very high concentration of NaCl could initiate this process.
Reversible precipitation could result from the protein being at a fairly high concentration while at or near its isoelectric point, or more generally, at a concentration in excess of its solubility under the conditions. Returning the protein to the original conditions, diluting it, or changing the pH would restore the solubility of the protein.
Dear Amulya Ramakrishna
Why did you added 5mM beta mercapthoethanol?
since in case you have S-S bond the addiction of reducing agent can induce protein unfolding.
which is the pI of your protein?
Did your protein contain free cysteines?
If not suggest to you to do not add reducing agent to preserve S-s bonds if presents and use desalting instead dialysis (which is long process)
you can find some more information about desalting on the following links
https://www.blogger.com/video.g?token=AD6v5dzeX21U_Er6jBChQIhE5zWtQhNjRjNXRPR7qk7KfWWSQxhf8PbUn4R6KLL0pn_7tU92M0-B3kEUHO9IkTpelmdO79QR53uSg29ZFV3j5D-siHAamTu1y2wOEQ8S4As_wpxzLvA
https://www.blogger.com/video.g?token=AD6v5dwNlVHz3BclDT9RLY-6KbEEqkS3RnRdHn4BqTsZW4j_prf3KZwHDMTzRYT-B9Yifvmde7RdExL5AArLFtg1Q13fu4-83o3M8bCka1jJ5PfWFK8B53BOpx6YrzvLr2qDiPgT15c
avaialble on my blog (ProteoCool)
best
Manueie
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