My protein (0.7ug/ul) is precipitating and becoming cloudy, once I add it to the quartz cuvette for UV fluorescence experiment while control protein GST (of the same concentration) is not precipitating. Also, the protein is not precipitating while in eppendorf and in room temperature. The cuvette is thoroughly washed and then used. My protein is in 20mM Tris, pH 7.5 and 50mM NaCl buffer. What changes shall I do to prevent my protein from precipitating?
Hi Divya,
Some questions : how did you come to use GST as control protein in your experiment? Is it similar in IP and activity to your assay protein? Did you test different concentrations?
Several suggestions :
1) Check if that it is your protein that precipitates and not salt (centrifugation and A280nm mesurement) 2). 20mM Tris concentration is not so strong, so that pH could change according to what you add to the cuvet in your experimental protocole (also, take care of pH variation of Tris buffers according to temperatuer) 3) Your protein may react with a component in your experimental protocole. 4) Depending on what type of protein you studdy, it may be sensitive to glass surfaces (may happen if it is activated at interfaces such as lipases for example).
Good luck
How do you wash cuvette?
Wash with acid, rinse in ultrapure water and then clean with acetone. Let it air dry.
Thank you very much Patrick Rouimi for your suggestions, I shall definitely check these. I had checked different concentrations and I took GST as my protein is also GST-tagged fusion protein. Regarding sensitivity to the glass surface, I want to know how can I test that? I just know my protein binds with Phosphatidylinositol phosphate. Can it have any problem related to this property?
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