A Splicing factor Protein that is prone to aggregation at/ above 0.5 mg/ml concentration. It also showed temperature dependent aggregation above 65 C. I need to do some Near UV CD studies, as well as DSC and ITC. So I need concentrated protein (approx 1 mg/ml). Buffer that I am using is (150 mM NaCl, 10 mM Tris Cl (pH 8.0), 40% Glycerol). For experiments Glycerol was removed by dialysis as it was interfering with CD data (Far UV). The protein has his-tag, and was expressed using IPTG in C41 cells.
I looked all of your explanation.I think if you set your pH value and the salt concentration(for his-tags) better,I hope you will solve this problem. Besides if you need mix the enzyme, you can mix it so gently. Because you use 40% Glycerol and please don't forget it has an vizcosity.
His tagged proteins do have a tendency of aggregation at high concentrations. First remove you glycerol. Glycerol helps long term stability but ups your chance of aggregation problems. Also, try to keep the pH at pI, or near whatever the protein eluted from your his column (his tagged proteins don't handle pH changes very well). Next substitute out your tris for a PBS, HEPES, or other physiological buffer at ph=7.0, again try to adapt your pH to where ever your protein is most stable (ie if your protein likes pH=4 try a buffering at 6.5, vice versa it likes pH=9 try buffering at 7.5). Next in order, try higher salt (0.5-1.5M NaCl), sugar (trehalose, glucose, etc), and lastly some mild detergent (0.05%-1% saponin, tween , triton). In my experience the corrected pH and the high salt are likely to work.
Try higher and lower salt concentrations and varying the pH within the physiological range.
Try change the solvent concentration and PH you can solve the problems.
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