Hi, We have a case where a 260 KDa protein crystallized at 3 mg/ml, that was the highest concentration we could achieve, without playing with the protein buffer. Protein-to-well-solution ratio was 3:1. When screening, we tested all ratios between 4:1 and 1:1. Crystals only appeared at 3:1 ratio. That is another important variable to test, I guess. Regards, -Andre.
________________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp (Hofkristallrat a.D.) [hofkristall...@gmail.com] Sent: Monday, February 27, 2012 1:25 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Desalting columns Why, in the first place, do you feel an urge to concentrate your protein above 3 mg/ml ? For crystallization, the concentration needs to be a) high enough to achieve supersaturation, meaning close enough to the maximum solubility in a given buffer so that the precipitant can drive the system in to supersaturation, preferably of a level where homogenous nucleation can occur (or you micro-seed, if necessary) b) high enough that sufficient material for crystals of acceptable size to grow is in the drop, which is generally the case, lest micro-crystal showers happen. There is ample evidence for proteins crystallizing below 3 mg/ml. The often quoted PDB/BMCD average of somewhere around 10 mg/ml is biased towards highly soluble, smaller (lower hanging fruit) proteins. Sometimes the shape of a distribution matters ;-) BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sangeetha Vedula Sent: Monday, February 27, 2012 8:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Desalting columns Dear bb users, 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. Thanks a lot! Best regards, Sangeetha.
