Jonas, scaling up may be tricky as the increased protein concentration affects the elution profile. Personally, I tend to think that scaling up may often be avoided by simply running the protocol that you have developed several times. Maximum recommended loading capacity of a 1mL monoS is 25 mg, do you actually need to go much higher? If you can get 10mg of protein from a single purification, you are in great shape.
As for resolution, the most important factor in my experience is not how shallow the gradient is (although it matters), but the flow rate. Some of them columns have specs that suggest up to 2ml/min flow rates, so naturally we tend to run them fast (getting 1 column volume per minute sounds so cool). But the resolution suffers - I may even go as far as to say that running the monoQ and such at 1ml/min means you are not taking advantage of its high resolution at all. I have seen in the past high-res ion-exchange columns do miraculous things when you run them at 0.1ml/min. Perhaps you also want to take a look at UnoS from Biorad. They are just as good as monoS (if not better), but are somewhat cheaper. Good luck, Ed. On Tue, 2011-01-25 at 20:06 +0000, Jonas Boehringer wrote: > Dear All, > I am currently looking into various cation exchange resins for the > separation of mono-, di-, tri-, tetra-,... ubiquitin. So far I have > been using a small Mono S column but would ideally like to both scale > up and increase resolution. Before I started searching for this online > I have never heard of the above resins and was wondering whether > anybody on this list has any experience, maybe even comparative, with > anything other than Mono beads (which are really expensive as well). > > > Many thanks in advance and best wishes, > Jonas > ---------------------------------------- > Jonas Boehringer > Department of Biochemistry > University of Oxford > South Parks Rd > Oxford OX1 3QU > United Kingdom > > > > >
