I've tried to purify recombinant protein from E.coli and I got a good single band in small-scale while several unspecific bands presented in LARGE-SCALE. I tend to Re-Purify the targeted protein (which still in Elution buffer that contain 250 mM imidazole) using Ni-NTA agarose gel.
Should I exchange elution buffer into PBS before Re purify the protein?
Your protein is not going to bind to the column if in elution buffer. So you need to either exchange it (or dilute it) in order to get binding a second time.
However maybe if you washed the column better in the large scale process you would remove the nonspecific bands.
Dear Asem Alsharef
it depends from which second purification step would you like to apply.
For example if you will run a SEC as 3nd step you do not need to exchange buffer since the SEC itself is able to do it (the protein at the end will exit in the buffer used for coloumn equilibration)
On the contrary for example if you would like to perform ionic exchange as 2nd step you need to buffer exchange your protein in a buffer that do not contain NaCl and a pH with the right value for allow the protein to bind to the coloumn.
best regards
Manuele
Ni-NTA chromatography is "bad" affinity system, so for best results you need to "overload" the column.
By doing this your protein will compete off non-specifics leaving you with a nice clean band.
This is why it's common for test purifications to look great and at scale bad.
However, if the other bands are degradation products they will stay present in the elution.
Depending on your elution volume you could change buffers rapidly with a HiPrep Desalting/PD10 columns or you could diafiltrate if your protein doesn't mind being concentrated.
DEar Asem Alsharef
if the protein is already pure, you do not have to re-purify it but you can just do buffer exchange using dialysis or desalting
you can find an overview of the different approaches for buffer exchange in the following link on my blog, ProteoCool:
https://www.blogger.com/video.g?token=AD6v5dzeX21U_Er6jBChQIhE5zWtQhNjRjNXRPR7qk7KfWWSQxhf8PbUn4R6KLL0pn_7tU92M0-B3kEUHO9IkTpelmdO79QR53uSg29ZFV3j5D-siHAamTu1y2wOEQ8S4As_wpxzLvA
best
Manuele
The contaminating proteins were present in the small-scale prep, just at a scaled ratio making them hard to see until to increased the prep size. If you have access to SEC this is the shortest path to both desalt and polish your protein. It all depends on the size of the other contaminants. May need to add an IEX step to refine. I agree with others, not much to gain from just rebinding to Ni-NTA, but if you wanted to you simply need to dilute the imidazole down to a concentration that is permissive to rebinding.
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