I am trying to crystallize a ~320 kDa protein that crashes out if
concentrated past about 3 mg/mL.
I would like to try to exchange it into various buffer-salt-additive
combinations to see which buffer works. For a starting point, I'd like to
use desalting colums.
Does anyone have suggestions for good buffer exchange and sample recovery?
I woud like to load about 250 uL onto each column.
~ 1 ml spin columns with Sephadex G25 or Biogel P6. Typically, when loading
200 ul of the sample onto 1 ml column, desalting efficiency is ~ 95% and
protein recovery is ~ 90-95% without volume change. Lowering sample volume
will increasing dealting and reduce yield but I never had the yeild worse
than 75% even with 50 ul volume.
Bio-Rad sells prepacked "Bio-Spin P-6" or you can buy empty columns and dry
P6 (by "fine" grade in this case) and pack to 1.2 ml per column, which will
take your 250 ul volume and still result in decent desalting. Pharmacia
probably sells something similar and many companies sell 0.5 ml spin
columns. I like P6 (polyacrylamide) better than Sephadex (dextran) because
usually (but not always) non-specific binding is lower.
- Dima
Thanks a lot!
Best regards,
Sangeetha.
