Am dealing with gold nanoparticles passivated with a mix matrix of (70% CVVVT-ol+30%Polyethylene glycol (PEG)). mixed with 0.3%, 0.5% 1% and 5% of Azide his tag (CVVVTAAHHHHHHK(N3) and (CVVVTAAK(N3)HHHHHH ) separately. the samples left overnight on the rotated wheel and then free ligand removed by five times centrifugation for 7 mins 10000 RPM with nanosep filter 10KD, then samples loaded to nickel column. Histidine should be stick to the column. in my case it didn't stick. I would be grateful if anybody could help and thank you in advance.
regards
Zaid Alghrair
If that's a protein-grade chromatography resin you're using, I'd view it as likely that the nanoparticles are too big. They need to get into the pores inside your nickel beads.
Contrary to certain widely circulated drawings, almost all the relevant affinity chemistry happens on the inside of the chromatography beads, not on the outside. That's because the inside surface of the highly porous beads is much larger than the outside. If something is too large to get inside (e.g. plasmids being a common source of surprise), the process will be so compromised, you usually get nothing at all.
So go ahead and compare the size of your nanoparticles with the size exclusion limit of your nickel resin. If they can't fit in, you know that's why.
