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His-tag protein purification, contamination issue

His-tag protein purification, contamination issue - Recombinant Protein Forum

His-tag protein purification, contamination issue - Recombinant Protein Forum. Discuss purification, expression, and isolation of recombinant and cloned proteins here.


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Old 03-10-2011, 02:12 AM
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Default His-tag protein purification, contamination issue



Hi everyone

I'm new on his-tagged protein purification. I'm working with a recombinant protein of 13.1 kDa expressed in Pichia and I'm trying to purify it with a Ni-NTA column according with Probond native protocol of invitrogen.
I run my Tricine-SDS-PAGE to analyse the sample after pass it trought the column, and the washing (I'm performing 6 washes with native washing buffer with imidazole 20 mM an pH 8.0), and the elutions (my elution buffer has 250 mM imidazole and pH 8.0).
The problem is that apparently I've a good recovery of my recombinant protein in the elutions but also got a little contamination with proteins of lower molecular weight. Usualy when I'm passing the sample trought the column, I work on it in a room at 4 C degrees.

Does anyone a suggestion to get better results?. I'm thinking on dialyzing my elutions against binding buffer and try to re-purify it because but I'm affraid of lose a lot of it and I must have my pure protein as soon as possible.

Does anyone experience something similar?

Thank you

P.D. Excuse my bad english.
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Old 04-07-2011, 01:16 AM
Pipette Filler
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Default Re: His-tag protein purification, contamination issue

Try doing a step gradient elution. For example, do a round of elution with 50mM imidiazole, then 150, then 250. It's likely that the contaminant proteins will get "knocked off" at different imidazole concentrations. You will want to elute at each concentration until no more protein comes off. It's best to test your samples as you're eluting...I usually take small samples (10ul) and put it into 100ul of bradford's reagent. By doing this through the elution process you can produce a nice chromatogram to see where your protein elutes best at, and how much you're getting.

Good luck

Quote:
Originally Posted by laurakjim View Post
Hi everyone

I'm new on his-tagged protein purification. I'm working with a recombinant protein of 13.1 kDa expressed in Pichia and I'm trying to purify it with a Ni-NTA column according with Probond native protocol of invitrogen.
I run my Tricine-SDS-PAGE to analyse the sample after pass it trought the column, and the washing (I'm performing 6 washes with native washing buffer with imidazole 20 mM an pH 8.0), and the elutions (my elution buffer has 250 mM imidazole and pH 8.0).
The problem is that apparently I've a good recovery of my recombinant protein in the elutions but also got a little contamination with proteins of lower molecular weight. Usualy when I'm passing the sample trought the column, I work on it in a room at 4 C degrees.

Does anyone a suggestion to get better results?. I'm thinking on dialyzing my elutions against binding buffer and try to re-purify it because but I'm affraid of lose a lot of it and I must have my pure protein as soon as possible.

Does anyone experience something similar?

Thank you

P.D. Excuse my bad english.
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