In general, I use about 5 mL buffer per gram of pellet, which seems in line with your usual standards. I would suspect that is a starting point of your colleagues problems. In practice, I weigh my bacterial pellet after centrifugation to ensure an accurate measurement. Bacterial pellet volume can vary depending on the media used (i.e. auto-induction media will produce increased cell density), as well as the protein expressed (i.e. toxicity of the target protein leading to cell death and decreased density in the culture).
Kelly Daughtry ******************************************************* Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 ******************************************************* On Thu, Oct 25, 2012 at 8:15 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 > > >
