purification failed - how to restart? - Protein diluted in many fractions (Apr/24/2008 )

Hi,
I tried to purify the his-tag protein from insect inclusion bodies on Ni-NTA.

First I tried with a bit strange protocol of the protein producer:

I solubilized the pellet in
1. Tris-HCL pH 7. 4, 0.2M NaCl, 2%SDS. (Just by pipetting in and out several time at RT).

Than I purified - (washing buffer: 20 mM Imidazol, elution buffer :20 mM Sodium Phosphate, 0.5 M NaCl, 400 mM Imidazol),

Most of the protein was in flow-through fraction, but a little also in elution fraction
I have freezed all fractions.


2. I tried to solubilze in 6M Urea, 20mM Tris-HCl pH 7.4, 0.5M NaCl. Also just pipetting.

This time I used washing and elution buffers the same as solubilizing (urea, etc), just with 20 mM and 400 mM Imidazole respectively.

Purification - most of the protein in flow -through fraction.
Freezed all fractions.

As a protein is "kind gift" from somebody, I want to redo the purification from all the fractions I have.

I think about the dialysis of all fractions in the following buffer:

20 mM Tris-HCl, 0.5 M NaCl, 8M Urea.

And than just load onto the column. To wash and elute with urea.

What do you think?

Thank you in advance for your help

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Usually when you have a his tag protein, use the agarose Ni NTA will be sufficient to purify it. Not necessary dialysis. Have you check if your protein is soluble or not? Like running on the SDS PAGE? Probably the protein still stuck at the inclusion bodies since it is harder to lyse inclusion bodies.

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Mayby I have not explained well the idea. I need to re-purify the same protein preparation, which is dispersed now in many fraction with SDS, Urea and different concentration of Imidazol. How to return to the point zero with all this? and to re-begin the NINTA purification?

Thanks!

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I think your problem was with using 2% SDS ( recommended for Ni-NTA resin - only 0.3%) So your protein was in micelles and don't bind to resin.

So the strategy is following it will be good to combine all fractions , dialyze against 6 or 8M urea solution to remove NaCl ( or decrease to 0,15 M ) and perform anion or better cation IEC. So you will solve two problems - remove SDS ( dialyse is not the way to remove SDS micelles ) and purify your protein . It will be better to know pI of your protein to choose IEC conditions correctly. If purity still not sufficient apply fractions to Ni-NTA ( but know without SDS).

Sorry I see that you dissolve your IB in urea without SDS second time and still your protein in fall-through fraction? So I think may be problems with Tag during preparation IB ( hyper sonication may be ) In this case only one way change purification strategy - use IEC in 6M urea as I wrote above)

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Merci! Ogromnoe spasibo! (I am Ukrainian, so I can speak Russian)

I will do surely cationic IEC.
Never done it before, so I have a stupid question: what properties of protein decides is this a cationic of anionic column?

Protein IP is 6.66

And if somebody be so kind to give me a IEC protocol, I will be very grateful!

Thankx

Alesia

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I have founded in my lab the DEAE Ceramic HyperD F columns (weak anion exchanger) made by PALL. I will try to use this, with the buffer of pH 8.0, for my protein which IP is 6.6.

I will load the protein in 8M Urea, 20 mM Tris pH 8.0, and will elute with the buffer 20 mM Tris pH 8.0, 0.5 M NaCl.

My question is: should I add Urea also to elution buffer?

Thank you!

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Well, after some research, I founded that i cannot use the anionic column, because SDS is negatively charged as well and will stick to the anion exchanger.

Correct me please, if I am wrong

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Privet Alesia!
Ochen rad uslyshat' rodnye golosa!

You are quite right ! It will be be better to use cation exchange column for ex SP or CM sepharose So SDS will pass through the column

Good luck!

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