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

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Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University