What
protein concentration are you using in your screen? It sounds like you
need to try lower protein concentration, or a screen with lower precipitant
concentration, or both. Hampton makes a Crystal Screen Lite with the
precipitant concentrations half of what they are in the normal screen (or you
can dilute the regular screen). Don't forget that protein concentration is
one of the important variables to optimize. For some complexes, I have had
best results with protein concentration as low as 2.5-3
mg/mL.
Evette
Evette S. Radisky,
Ph.D.
Assistant
Professor and Associate Consultant II
Mayo Clinic Cancer Center
Griffin Cancer Research Building, Rm
310
4500 San Pablo
Road
Jacksonville, FL
32224
(904) 953-6372
(office)
(904)
953-0046
(lab)
Dear CCP4 Community,
My apologies for the off topic question to the bb.
I am trying to crystallise a small protein-protein
complex. I purify as a complex protein after expression in BL21DE3 cells
through NI-NTA affinity chromatography. pI of one of the protein
is around 10.6 where as another component have 4.0. I am in the
screening stage of the crystallisation. During crystallistion process
it precipitate very quickly as i add the protein into the
crytallisation solution drop. This happens in almost all the condition of the
hampton INDEX and crystal screens. I purify this complex in Tris-Cl buffer
at pH=8.0. it is happily soluble and donot prcipitate at this pH in the
same buffer. I can concetrate this protein upto ~10-15mg/ml. could any
one suggest the solution, that will be most appreciatable.
Thanks in advance
Ron
