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protein solution cloudy after dialysis


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#1 GSUOC

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Posted 17 August 2011 - 07:26 PM

Hello,

My purified protein is cloudy after dialysis. Is there any way to make it clear solution, so that it does not interfere in my crystallization experiments. Does any body had this problem?

#2 Adrian K

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Posted 17 August 2011 - 07:34 PM

I think it had to do with your buffer's ph... did you check your protein's PI?
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#3 GSUOC

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Posted 18 August 2011 - 09:15 AM

I think it had to do with your buffer's ph... did you check your protein's PI?

Yeah my protein's PI is 6.95 and the buffer i used is of ph 7.8.

#4 mdfenko

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Posted 18 August 2011 - 11:18 AM

is your protein insoluble in the presence or absence of salt (e.g.- actin polymerizes in the presence of salt, myosin is insoluble without salt) or some other additive (detergent,...)?

Edited by mdfenko, 18 August 2011 - 11:19 AM.

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#5 GSUOC

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Posted 18 August 2011 - 01:59 PM

is your protein insoluble in the presence or absence of salt (e.g.- actin polymerizes in the presence of salt, myosin is insoluble without salt) or some other additive (detergent,...)?


I precipitated with ammonium sulfate and used the buffer of 5mM 2-ME and 10mMTris HCl of ph 7.8
Would the amount of protein make it look cloudy? Because, other fraction of the same protein from FPLC is clear. I did not pool the fractions from the protein peak.

#6 mdfenko

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Posted 19 August 2011 - 11:49 AM

I precipitated with ammonium sulfate and used the buffer of 5mM 2-ME and 10mMTris HCl of ph 7.8
Would the amount of protein make it look cloudy? Because, other fraction of the same protein from FPLC is clear. I did not pool the fractions from the protein peak.

do you precipitate from the same buffer that you use to resuspend?

some concentrated proteins will be cloudy, but, cloudiness often indicates that the protein is not fully solubilized (or aggregated).

it's possible that the cloudiness is due to residual ammonium sulfate and dialysis will clear it.

in which buffer do you run fplc (gel filtration?)? if it is gel filtration then you need to maintain an ionic strength of ~0.2M to prevent non-specific binding to the matrix.
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