i am concentrating my protein for crystallization using amicon ultra.the protein is in 50mM tris,5% glycerol and 50mM KCl.every time i get precipitate at the membrane.the membrane becomes yellowish and when i pipette protein out i find precipitate.ultimately i get upto 7-8mg/ml.but i need at least 20mg/ml.
can anyone help me out plz?
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#2
mdfenko
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Posted 15 July 2009 - 07:59 AM
some proteins will aggregate when concentrated beyond a certain point.
sometimes you can add something to the solution that will prevent aggregation or, at least, shift the point up.
we use peg-8000 to allow us to concentrate our proteins. the peg co-concentrates on the membrane we use (ym-10) so we start with a low concentration and the final concentration will be about 1% (not more than 5%). we also find that peg can sometimes disaggregate the proteins.
sometimes you can add something to the solution that will prevent aggregation or, at least, shift the point up.
we use peg-8000 to allow us to concentrate our proteins. the peg co-concentrates on the membrane we use (ym-10) so we start with a low concentration and the final concentration will be about 1% (not more than 5%). we also find that peg can sometimes disaggregate the proteins.
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#3
DaveD
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Posted 11 February 2010 - 09:44 PM
What may be happening is that you're concentrating the protein to a concentration that's above its solubility in the buffer system you're using. Changing salt concentration, optimize pH and adding additives are typical ways to increase a protein's solubility.
The OptiSol protein solubility screening kit does that for you in one experiment. It's got 90 different buffers with systematically changed pH, additive, different salts etc. Once you've run it you have defined the 'sweet spot' of your protein.
If you're lucky they'll sign you up as a beta tester and you get to use a kit for free.
DaveD, My 2 ct
The OptiSol protein solubility screening kit does that for you in one experiment. It's got 90 different buffers with systematically changed pH, additive, different salts etc. Once you've run it you have defined the 'sweet spot' of your protein.
If you're lucky they'll sign you up as a beta tester and you get to use a kit for free.
DaveD, My 2 ct
mdfenko, on Jul 15 2009, 07:59 AM, said:
some proteins will aggregate when concentrated beyond a certain point.
sometimes you can add something to the solution that will prevent aggregation or, at least, shift the point up.
we use peg-8000 to allow us to concentrate our proteins. the peg co-concentrates on the membrane we use (ym-10) so we start with a low concentration and the final concentration will be about 1% (not more than 5%). we also find that peg can sometimes disaggregate the proteins.
sometimes you can add something to the solution that will prevent aggregation or, at least, shift the point up.
we use peg-8000 to allow us to concentrate our proteins. the peg co-concentrates on the membrane we use (ym-10) so we start with a low concentration and the final concentration will be about 1% (not more than 5%). we also find that peg can sometimes disaggregate the proteins.
