Originally Posted by cali
I'm working with a Gram- bacteria. I did a cell fractionation to separate priplasmic, cytoplasmic and membrane proteins. The membrane protein pellet should contain only insoluble proteins but when I resuspend it in PBS and do a slow centrifugation (4000 xg), I have proteins in the supernatant encompassing my unknown protein of interest! I'm not using detergent to solubilize them. Does anyone have an idea of what these proteins might be??
Thanks for your help!
How about a greater centrifugation speed, I'd do 14,000xg 10 min at 4C.
4000xg is a force and will knock down proteins based on size while the buffer will enhance or undermine the insolubility of the protein(s). Greater force will be needed to knock down smaller proteins (plus the whole insolubility may change if peptide chains break off). It's unlikely that 100% of the protein is insoluble.
Though you have an insoluble protein fraction, you have to realize that solubility is dependent on the solution around it. The protein or proteins may become more soluble
when resuspended in PBS (a different medium).