I have my purified proteins in a solution containing 37% PEG 8000. I tried to precipitate the proteins by using TCA but it doesn't work. Does anyone know how to concentrate or precipitate my proteins to be loaded in an SDS-PAGE?
Dear Francisco,
Two ideas:
1) increase PEG concentration until your protein precipitates, spinning it down would allow you to have a pellet that can be dissolved in Laemli buffer
2) try a methanol/chloroform precipitation which is a phase partitioning process, could work but never tried myself with PEG
Good luck!
Dear Didier, thanks very much.
Do you add PEG from a concentrated solution to the protein solution? How do you test that the protein is precipitated? Do you precipitate it al 4ºC? For how long?
Have you ever tested if PEG interferes with analysing your proteins by Western-blot?
Dear Francisco,
For small volumes it's probably easier to add a concentrated PEG solution. Usually the bigger the MW of the PEG the less you need to precipitate, the two most common that I use are PEG 6000 or PEG 8000. You can visually check that precipitation is effective by the appearance of cloudiness, only works for concentrated protein solutions (>1mg/ml), otherwise an activity test and/or gel electrophoresis of the pellet you obtained could be used.
To my knowledge PEG will not interfere with downstream WB, most of the PEG remains in the supernatant and there's very little carry-over to your gel.
Hope this helps.
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