Agarose bead size in affinity chromatography - (Oct/14/2013 )

Hello everybody,

I have a quick question regarding agarose bead size in affinity chromatography, specifically nickel-coated beads for purification of his-tagged proteins. I'm using small columns (1 ml) on an FPLC. If I have a selection of the same column size with the same binding affinity, only differing in bead size (i.e. 34 µm or 165 µm), what is the advantage of smaller beads? I know that larger beads give me a higher possible flow-rate (less back-pressure) which is generally a good thing, as it saves time. OK. But the smaller beads? Binding capacity is the same (as stated by the manufacturer), resolution should not matter either, right? As this isn't a "dumb" elution by time (as in size exclusion chromatography) but an elution with Imidazole in which I can determine the exact amount to add to specifically elute my protein of interest empirically, so the resolution should be the same no matter the bead size.

So, anyone using columns with small beads on purpose - if so, why?

Thanks!

map3k

I use GE's HisTrap resins and they offer a HP version (bead size 34um) and a FF version (bead size 90um).  I use the FF as it suits my needs fine but the smaller bead size of HP does provide slightly better capacity and resolution.  Resolution can still be obtained even though it's not a 'dumb' elution.  Using an automatic purifier, like an AKTA, a gradient elution can be done to help resolve the protein of interest from contaminating host cell proteins that bind with slightly different affinities so the smaller bead size does hold some value if you've got the tools to make use of it and need the better resolution.....

Thanks! Yes, that does seem logical. I'm pretty much in the same situation as you i.e. the FF suits my needs in 90% of all cases but I might just have a go at the HP for some of the more stubborn samples where I still have problems with purity. Will try a really shallow gradient and check if I see significant differences...