My interested protein size is 36 kda. After protein purification by Ni, there is extra band about 60 kda. I have tried to eliminate this contaminate by several washing time but it didn’t disappear so thought it would be ok if I use amicon Millipore 50kda. I guessed the concentrated protein on filter are greater than 50 kda and I can find my protein without always contaminate in flow through. Unfortunately when I run the filtrated and un filtrate protein, see there is no band in flow through and my protein and its extra 60 kda proteins all are as retained protein. I am confused what is the problem.do amicon millipore filters 50 kda retain 30 kd proteins? How can I get rid of contaminate?
I should emphasize that the amicon millipre 15 ml 50 kda has been used.
thank you
Hi there,
Does your protein oligomerize under native condition? If so it is normal for oligomers to be retained by 50kD cut-off membrane as the simplest oligomer (dimer) size would be 72kD. To answer your question about membrane cut-offs, they are approximative and calculated for globular protein model.
No, these filters aren't intended for reliable size-based purification. They're mostly used for buffer changes (separating things with very large size differences). What you're describing might be done reliably using size-exclusion column.
Although you should also be able to tweak your affinity column to get rid of the band. E.g. if it's an aggregate, change pH, salt, or reducing condition to prevent it. Or, if it's an endogenous protein, increase imidazole during washing, or change the metal ion.
It is not advisable to use spin columns to remove contaminants from purification. If you are unable to remove this contaminant during purification through Ni resin (which is not surprising as non-specific binding is common), then I would recommend you add an additional purification step (IE try an anion exchange column, one of if not the most common steps for proteins purified under physiological buffer conditions pH 6.5-8).
If you do not have access to an HPLC or AKTA FPLC for protein purification a variety of ion exchange resins are available that would be suitable for gravity flow applications (http://www.spectrumlabs.com/chrom/IonExchange.html). Utilize these, and varying concentrations of buffer salt to separate away your protein of interest from other contaminants.
Hi,
It is not advisable to purify the protein through amicon tubes, because it just used to concentrate your protein in the salt solution. So, it is suggested to purify your protein through size exclusion column or FPLC.
Best of Luck
Nilesh
It has been pointed out already that the ultrafiltration devices are not intended for purification, thus I will adress the other problem.
In my opinion, you are not able your POI from the contaminate because they interact. Thus even size exclusion chromatography will not work (but of course it would be good to try, if you can). I bet you expressed your protein in E. coli and the contaminant is some chaperon.
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