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His-tag protein purification


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#1 deep1985

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Posted 16 January 2011 - 11:03 PM

Hi everyone.

Recently i purified a his-tag protein(34kd) using Ni-column, and run the samples through AKTS(superdex 75) to obtain pure protein,

and then i concentrated the protein to 3mg/ml using a magnetic filter. After concentrating the protein, i stored the protein at 4degrees, i noticed that the protein got aggregated in the solution. Can anybody suggest the reason behind it? and what can i do to avoid it?

ThankYou
Deep

#2 deep1985

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Posted 16 January 2011 - 11:06 PM

Hi everyone.

Recently i purified a his-tag protein(34kd) using Ni-column, and run the samples through AKTS(superdex 75) to obtain pure protein,

and then i concentrated the protein to 3mg/ml using a magnetic filter. After concentrating the protein, i stored the protein at 4degrees, i noticed that the protein got aggregated in the solution. Can anybody suggest the reason behind it? and what can i do to avoid it?

ThankYou
Deep

Adding a note:

AKTA is a size exclusion chromatography column..

Thank you

#3 protolder

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Posted 16 January 2011 - 11:51 PM

Hola, At the same time that you concentrate, has to change the buffer to some adecuate for your protein, that I suposse you do it. Anyway there are in the market some stabilizing cocktails for pure protein solutions as the Pierce´s. If your protein is commercial check in the supplier by the storage buffer or for the one for any similar protein. Buena suerte

#4 deep1985

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Posted 17 January 2011 - 11:32 AM

Thank You so much for the reply...will check it out...

deep

#5 mdfenko

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Posted 19 January 2011 - 08:38 AM

some proteins tend to aggregate when concentrated above a critical concentration.

certain additives may prevent or even reverse aggregation. you can try 1-2% ethylene glycol, 1-5% polyethylene glycol, high salt, low salt, different pH (may be too close to pI), some detergents (chaps, triton, deoxycholate,...).
talent does what it can
genius does what it must
i do what i get paid to do

#6 deep1985

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Posted 19 January 2011 - 02:08 PM

some proteins tend to aggregate when concentrated above a critical concentration.

certain additives may prevent or even reverse aggregation. you can try 1-2% ethylene glycol, 1-5% polyethylene glycol, high salt, low salt, different pH (may be too close to pI), some detergents (chaps, triton, deoxycholate,...).



yes...definitely will try some conditions mentioned..

Thank You so much...that was really helpful.
Deeep




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