precipitation of protein with acetone - (Oct/27/2008 )
I am precipitating my protein with acetone, because the dilution is too high for SDS-PAGE. It precipitates all right, but there's a problem with resuspending the pellet. The solution is not clear, the protein isn't really dissolved and Bradford assay doesn't show me any increase in concentration (it should be around tenfold - 1,5 ml extract resuspended in 150 microliter of the buffer).
What should be the buffer composition to dissolve it, measure the concentration with Bradford (either that or with a different method), and then do a western? Should I heat it or add something else?
What should be the buffer composition to dissolve it, measure the concentration with Bradford (either that or with a different method), and then do a western? Should I heat it or add something else?
Um, again it seems the question answered itself. I'm just using another technique...
What should be the buffer composition to dissolve it, measure the concentration with Bradford (either that or with a different method), and then do a western? Should I heat it or add something else?
Um, again it seems the question answered itself. I'm just using another technique...
I am also having the same problem of low protein concentration. Can u suggest me a method to overcome this?
Thank you!!
Add 200 ul of 1% SDS to the acetone precipitated protein pellet, pipette and vortex to fully suspend the pellet, then incubate at 90-100 °C for 3 minutes and you will completely re-solubilize the protein pellet. Good luck!