White precipitate after unfreezing from -20 degrees centigrade - (Mar/09/2007 )

hi all,

After His-Bind column purification, the purified sample (a clear solution without any precipetates) was stored in -20 degrees centigrade.

After 18 hours of storage, the sample was directly unfreezed in a water-bath (37 degrees centigrade).

It's so strange that a lot of white precipitaes appear in the sample.

The sample was stored in an eppendorf and without opening the cap.

So~what is the white precipitate?

Thanks for your help!

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may be your protein or a buffer component precipitated; what is the precise buffer composition?

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Do you desalt your protein before freezing? Try to measure protein conc in solution. So you can determine nature of youe sediment? If concentration decrease
may be your protein not properly refolded and so your protein aggregated and fall down.

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The content of sample buffer is: 500 mM imidozole, 20 mM phosphate buffer, and 0.5 M NaCl

The sample was not desalted before freezing.

However, the precipitaes do not appear in the same storage protocol in other His-tagged protein purification in our lab.

Another question is:
Does the decreasing temperature enhance salt-out proteins?



Thanks to "circlepoint" and "The Bearer" and whom answers the question

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yes, especially high-concentrated protein solutions but depends very much on the nature of protein; if it was eluted in its denaturized form, risk of precipitation strongly increases compared to the corresponding native protein

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