Hi Kavya,
You`r likely working with a very low PI sample, right? . Here are a few
suggestions
Final purification step at a pH closer to the pI:
Adjusting the pH during purification to be closer to the pI can reduce the
solubility and promote precipitation.
Non-PEG precipitants based kits:
Using precipitants like MPD, AmSO4 , or organic solvents can be effective in
inducing protein precipitation.
Adding organic solvents (e.g., 4% 1-propanol):
Organic solvents can disrupt the water structure around proteins, leading to
their precipitation.Make sure to carefully optimize the concentration of the
organic solvent to avoid denaturation or other adverse effects on the protein.
Glycosylation:
If your protein is glycosylated, this can indeed increasse solubility and
stability. Consider using enzymes like PNGase F to remove glycosylation if it's
not crucial for your experiments.
good luck
Andre S. Godoy, PhD
Universidade de São Paulo
Instituto de Física de São Carlos
Av. João Dagnone, 1100, Jd. Santa Angelina13563-120 - São Carlos, SP, Brazil
Em segunda-feira, 5 de fevereiro de 2024 às 07:27:28 BRT, kavyashreem
<[email protected]> escreveu:
Dear All,
Has anyone worked on a protein which is highly soluble even at 80mg/ml?
We have one such candidate, which does not precipitate even at 80mg/ml instead
forms phase separated globules in crystallization plate, which eventually
hardens over a period of 1 to 1.5 months (which is florescent under UV
microscope.)
We tried screening at different pH, but failed to get any hits.
Since we got few conditions in which the phase separated globules solidified,
we focused on them and expanded with 120mg/ml protein, still there were not
visible precipitates except for the phase separation. This has been a
challenging target so far. We have tried with different constructs, which
unfortunately are not soluble!
Does POMs help in such cases? Or do you have any other suggestion.
Thank you
Regards
Kavya
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