We resuspend in a low ionic strength buffer in a 2-3:1 ratio (mL/g). We typically get 15-25 g of wet packed cells per liter of TB medium, and resuspend in 40 mL of buffer. This goes straight into a bead beater for complete, gentle homogenization in 8 min. Protease inhibitors are optional. We purify quickly at 4 deg C.
Roger Rowlett On Oct 25, 2012 8:16 AM, "Raji Edayathumangalam" <r...@brandeis.edu> wrote: > Hello Everyone, > > Sorry for this rather naive and non-CCP4 question but I am very curious. > > My rule of thumb is to resuspend bacterial cell pellets in about 1-2% of > the original culture volume for a wet weight of about 3g of bacterial > pellet per L of culture volume. For example, Typically, the total volume > of my resuspension for a 6L-bacterial cell pellet is around 60-70mL or > about 40mL, if I really try to minimize the volume of buffer. Every > protocol I have read over the years seems to indicate something similar. > > In troubleshooting one of my colleague's protein preps, I found that she > is resuspending 6L of cell pellet with a total of pellet+buffer volume of > 5mL. In practice, I would not physically be able to resuspend a 6L pellet > in 5mL (3g pellet/L culture) without making a very viscous and lumpy soup. > My suspicion is that such small volumes are a source of some of her issues, > including a high number of impurities in her elution from affinity columns. > > I'm curious to hear what other folks do and recommend. > > Cheers, > Raji > > > -- > Raji Edayathumangalam > Instructor in Neurology, Harvard Medical School > Research Associate, Brigham and Women's Hospital > Visiting Research Scholar, Brandeis University > > >
