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 > <[email protected]> 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
