07 November 2019 3 5K Report

I am currently having difficulty optimizing a binding buffer for Ni-NTA protein purification under denaturing conditions. My starting buffer is currently composed of 100 mM NaH2PO4, 1 M NaCl, 10 mM Tris-HCl pH 8, 8 M urea, and 5 mM imidazole; however, I am having large loss of protein immediately after incubation with the resin (initial run-through of cleared lysate through the gravity column followed by washes with binding buffer). Can someone recommend a fast initial screening protocol, where I do not need to complete the entire protocol in order to test a new buffer? The protein I am currently working on is a soluble 27.2 kDA lipoprotein for reference. Thank you!

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