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Hi Nicola, One way to do it is to dilute your protein, 10-100 times, and add zinc (also diluted), then concentrate. Here is the procedure we used some time ago for a zinc-binding protein: “S100A12 was diluted to 0.1 mg/ml-1 (approximately 10 mM) in a buffer containing 20 mM Tris-HCl pH 7.5, 200 mM NaCl and 10 mM zinc-acetate and concentrated to 10 mg/ml by ultrafiltration with 10 kDa cutoff membrane (Amicon Ultra 15, Millipore; Vivaspin 500ul, Sartorius Stedim Biotech). The procedure was repeated three times to achieve complete saturation with zinc while avoiding aggregation due to higher zinc concentrations”. It worked, and we got a zinc complex :) Good luck, Olga > On 9 Dec 2018, at 21:31, Nicola Evans <[log in to unmask]> wrote: > > From a fluorescence scan it would appear a protein I am working on has zinc in it. The occupancy is likely to be very low however (a structural homologue has several zincs in the x-ray crystal data but at 0.5 occupancy), as there isn't anything obvious in the electron density map (perhaps some of the waters are zinc) and an anomalous difference map wasn't possible to obtain on our last beamtime. > > Ideally I would want to re-express the protein with zinc added to the culture conditions, but I am time-restained, so I was wondering if it is possible to add zinc to purified protein instead? I have heard it can cause proteins to crash out. I have quite a lot of protein frozen so I can try a few things. I would appreciate any advice on how much to add from anyone who has had success with this before? > > Thanks in advance! > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1