Could anyone suggest me a good protocol for purification of my his-tagged protein. I am eluting my N-terminus his-tagged protein with imidazole but after purification my protein has no more enzymatic activity. It could be due to imidazole inhibition which is reported in some cases that I have read. Could anyone suggest me a good protocol to elute my protein through the column using pH based elution.
Dear Muhammad
which kind of enzime are you producing? A metalloenzime?
Is it possible to do a buffer exchange to restore the activity? I would do this first. Also, Do you know if your protein will not precipitate at lower pH?
When the pH is dropped below 7 to 6 or so, the histidines become charged, and no longer bind the Ni NTA as well, so I believe people elute at a pH around 5.5 to 6. I would check the isoelectric point of the protein and make sure it will not precipitate at the lower pH values. The protocol can be found in the manufacturer"s directions with what resin you purchase.
If the protein activity were reported to be adversely affected, I would suggest to use a different affinity purification, such as amylose or GST..
You could do an elution with 5-10 mM EDTA which will strip off the metal and so release the His-tagged protein. However, some of the newer resins are more resistant to EDTA stripping so you may have to increase the EDTA concentration significantly (see spec. sheet for your resin).
use 0.1M glycine pH 3.5 for elution and neutralize with1M tris buffer
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