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For a complex that size, a quick and dirty way to check for aggregates might be to look at the samlpe under EM with negative stain. As far as getting a homogeneous protein solution, are there subunits that might have disordered domains/termini? If so maybe the clones can be engineered to express the stable domains. This is a good program to check for disorder in your protein: http://bioinf.cs.ucl.ac.uk/disopred/ And of course playing with the buffer(type, pH, salt, additives) might yield a preferred condition in which the protein is happy. Good luck, Kianoush > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > I am starting to work on a large protein complex ( about 1 mega dalton) > that requires some detergent during the last purification steps and > during protein concentration to keep it in solution. Even then, we were > only able to concentrate it to about 1 mg/ml. I just took over the > project and wanted to characterize the protein for solubility and DLS > under various buffer conditions. Looking at protein drops under the > microscope, I noticed that there is phase separation in the protein > solution even at 0.5 mg/ml protein. The protein is in 20 mM buffer, 0.25 > M KCl, 5% glycerol, 0.25% beta-octylglucoside. I will probably not be > able to measure DLS on such a sample and I expect it to be a problem for > crystallization as well. Any suggestions what to try to get a more > homogeneous protein solution. > > Thanks > > Ursula > >
