I typically use a 10:1 ratio of lysis buffer to paste (e.g. 100 ml for 10 g) for E. coli expression and lyse by high pressure homogenization (e.g. APV at 700-800 bar). I often add Benzonase to the lysate prior to clarification by sedimentation. This works great for highly expressed proteins with affinity capture as the first purification step.
For those who prefer to use really thick suspensions for lysis (like less than 5:1), you should keep in mind that your pellet after clarification will contain a significant amount of the added lysis buffer and, consequently, the protein that you are trying to purify. Hence, your yield will be lower. - John Sent from my iPad On Oct 25, 2012, at 8:15 AM, Raji Edayathumangalam <[email protected]> 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 > >
