Hi all.
My protein forms aggregates but I couldnt use any additives like glucose or glycerol because It forms oligomers.
Thank you
Did you try to sonicate the protein solution? It would be helpful to dissociate protein aggregates without additives. Be careful of keep chilled the protein solution using ice.
Another choice is to use ethanol up to 20%, but I don't know if that affect the protein of your interest.
Good Luck!
Are the oligomers formed in the presence of osmolytes defined (say, dimers or trimers)? If so, they may represent the natural state of your protein. Ultracentrifugation (use the Martin & Ames 1961 method if you don't have an analytical instrument) or light scattering should tell you that.
I didnt try it but I think The protein return to aggregate after the sonication, but Which sonication conditions do you use? The problem to add additives is They form differents Oligomers. Thank you.
Dear David:
Have you ever used detergents, glycine or glicerol? They may help.
Can you add a cation exchange chromatography step to your purification and elute fractions at increasing salt concentrations (then test fractions with something like SEC)? I think the aggregates will preferentially stick to the column, but you may need to bring down the pH and conductivity of your load.
Hi David,
you can add salt in the buffer solution to avoid the aggregation.
all the best
Hello David,
in my lab we use 6 M guanidine-HCl or 8 M urea, it solubilizes proteins and prevents big proportion of aggregation in our setting. I don't know is it compatible with your regime.
The aggregation you a facing may be due to several reasons-
Please check first whether it is a concentration dependent aggregation?
or Temperature dependent
or the pH of the buffer is more deviated than the Iso-electic point of the protein?
In first case you can keep your protein in diluted state (first you check at what conc. it is aggregating),
You can spin at 4k rpm for 5-10 min at 4C, take the dissolved protein and re-add buffer in the precipitate keep for o/n on rocker, it will dissolve again.
Most important is pH, you have to keep the buffer pH near PI of protein,
Sometimes changing buffer also helps, like if you useTris buffer pH 7.8, you can shift to Sod Phosphate buffer or HEPES of same pH,
changing salt composition / concentration will also help.
Actually, solubility of proteins is lowest at the pI, as the protein is uncharged and hence interaction with water is reduced. Unless you plan protein precipitation (e.g., with ammonium sulphate or acetone) your buffer should have a pH different from the pI of your protein.
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