9 replies to this topic

[precip_man]

I have a protein with a pI of 4.4 and at concentrations greater than 200uM (needed for NMR sample), it aggregates (although still soluble).  The current buffer is 25mM PO4-, 25mM KCl at pH 7.  What are some ways to help keep "unaggregated."  Thanks for your help.

Rolly

[mdfenko]

you may need to increase the salt

or you may want to try adding 1-5% peg 8000

or a little (i'm not sure how much) ethylene glycol

[DeeDee]

mdfenko, on 06 January 2011 - 01:30 PM, said:

you may need to increase the salt

or you may want to try adding 1-5% peg 8000

or a little (i'm not sure how much) ethylene glycol

Hello,
I had tried PEG and ethylene glycol to deal with soluble aggregates of my protein a few months back, but these seem to affect the enzyme activity of my protein.Also only PEG was effective in breaking the aggregates to some extent.Are there any other agents that can be used,as high salt concentration alone does not help much.Also,when I dialyse my protein out of 2M ammonium sulphate containing buffer into a OM ammonium sulphate buffer( with 500mM NaCl), my protein falls out.Does Ammonium sulphate stabilize a protein under certain conditions? I hope my questions are not intruding into this thread as I feel they are in the same area as the original question.

DeeDee.

[mdfenko]

is your protein soluble in 2M ammonium sulfate? or is it stored as insoluble precipitate? if it is stored as precipitate then the high sodium chloride may be maintaining the precipitate.

you can try a lower salt concentration (eg 0.1 or 0.2M).

what is the pI of the protein and the pH of the buffer? if they are too close to each other then the protein's solubility may be tenuous, at best. you may want to try another pH.

[DeeDee]

mdfenko, on 13 January 2011 - 10:45 AM, said:

is your protein soluble in 2M ammonium sulfate? or is it stored as insoluble precipitate? if it is stored as precipitate then the high sodium chloride may be maintaining the precipitate.

you can try a lower salt concentration (eg 0.1 or 0.2M).

what is the pI of the protein and the pH of the buffer? if they are too close to each other then the protein's solubility may be tenuous, at best. you may want to try another pH.

The pI of my protein is 5.1 and the pH of the buffer I use is 7.5. When I do a 0-55% Ammonium sulphate fractionation,I find my protein in the pellet.This pellet is then resuspended in 2M Ammonium sulphate containing buffer( with 100mM NaCl)and is passed through a phenyl sepharose coloumn where my protein elutes at 1.75-1.5M Ammonium sulphate concentration( sorry,2M in the previous post was a mistake).At this stage, the elution fractions are  clear (in the sense that I cannot comment upon soluble aggregates), there are no prcipitates.
When I dialyse these fractions to remove the ammonium sulphate against a O Ammonium sulphate buffer( with NaCl as salt), heavy ppt occurs.
Earlier we used 100mM NaCl when we observed the precipitation and we thought that a sudden shift from high to low salt levels was the problem so we raised the NaCl to 350mM once and 500mM the second time.In both cases heavy precipitation occured.
The 1.75M fractions however donot show any precipitate on storing for 4-5 days on ice.
Is there some way of solving this problem?

DeeDee.

[mdfenko]

i used to work with myosins. they require high salt (0.6M kcl, more effective than nacl) or atp (1-2mM, with 0.3M kcl) for solubility. you may want to try using kcl instead of nacl.

do you include ethylene glycol in your elution buffer for phenyl sepharose?

you may want to try 1-2% ethylene glycol in your dialysis buffer.

look at the conditions of your initial extraction and purification steps up to the ammonium sulfate precipitation. they may give you clues to the requirements to maintain solubility or to solubilize your protein.

Edited by mdfenko, 14 January 2011 - 07:00 AM.

[precip_man]

we have PEG 3350 in the lab...what is the difference between that and 8000?  Should i use ethylene glycol and PEG and high salt?

[mdfenko]

peg 3350 should also work (we use 8000 because it concentrates during ultrafiltration on 10 kDa membrane).

high salt with peg or ethylene glycol should be okay but we never tried peg and ethylene glycol in combination.

[precip_man]

so you mean you start with a very tiny amount of PEG so that it ends up being 1% in final concentration with protein?  

I cant seem to find out how PEG actually prevents aggregation, would you mind explaining?  Also would you recommend using chemicals like tween and NDSB for this as well, or in addition to to PEG and glycerol?

[mdfenko]

precip_man, on 02 February 2011 - 08:19 AM, said:

so you mean you start with a very tiny amount of PEG so that it ends up being 1% in final concentration with protein?  

yes. we have peg-8000 in solution at 0.05%. we concentrate the protein ~20x and the final peg concentration is ~1% (we found that we get stoichiometric recovery of our protein up to 20x concentration, your results may vary).

Quote

I cant seem to find out how PEG actually prevents aggregation, would you mind explaining?

forgive me, i don't know the actual mechanism. we found it worked when we tried concentrating our protein by dialysis against solid peg (powder) and went from there. that doesn't mean that the mechanism isn't known, just that i haven't looked.

Quote

Also would you recommend using chemicals like tween and NDSB for this as well, or in addition to to PEG and glycerol?

you can try them.

Edited by mdfenko, 02 February 2011 - 09:12 AM.