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I use a 10 ml GE-healthcare Ni-sepharose FF column, packed myself. I strip it with EDTA after a run and then recharge before the next one. It's has probably been used 30+ times, and I have no plans or repacking it anytime soon, it still works as good as it ever did. We purify secreted proteins from insect cells, so it's a pretty clean sample. If you use lysates I would imagine you would need more intensive cleaning with detergents and denaturants. But if the resin still turns blue (or pink for Co) it ought to work fine in my opinion, unless the frits are clogged, or a large amount of precipitation is trapped in the beads. I would unpack the column and wash the resin in batch mode in that case. Nat On Fri, Jul 23, 2010 at 1:20 AM, Artem Evdokimov <[log in to unmask]> wrote: > Hi, > > His-Select has been my favorite resin for IMAC for as long as Sigma's > been selling it. It's not the cheapest resin but at roughly $11/ml > packed bed volume and a capacity of ~10-20 mg protein per ml of resin > bed (depends on protein size, quality, and other factors) there seems > to be little point in regenerating the resin beyond 1-2 times. In > other terms, even at single-use it's still a good value since cutting > down on purification stages and duration (without sacrificing protein > quality!) is a certain way to increase useful protein yield and (in my > opinion) the likelihood of crystallization... > > I've also noticed that recycled resin gives somewhat more contaminants > (which is true for pretty much any brand of IMAC media) and since > 10-15mg of first-stage protein goes a long way in our lab I do not > feel particularly bad when I sacrifice a couple of ml of resin to be > sure that my protein is as good after primary separation as it can > ever be. > > Cobalt loading in my hands did not result in improved quality as the > resin binds very few contaminants by itself if used appropriately. > Primary contaminants arise from (a) sub-optimal coverage of binding > sites by the target protein and (b) from unwanted 'passengers' riding > on your protein of interest. The former can be minimized by dialing > the protein/resin ratio such that the protein is in slight excess > (i.e. resin can be saturated) and the latter can often be avoided by > proper selection of lysis and purification buffers (pH, ionic > strength, cosolutes, detergents and so forth). Your mileage will vary, > especially with membrane proteins or hideous sticky/aggregated > monsters like those that seem to frequent my lab way more often than I > like... > > The HF form of this resin is very convenient for semi-batch > separations (i.e. lysis and incubation with resin in batch, followed > by washing and elution on a column of some sort). This way a number of > proteins can be quickly purified in parallel without any special > equipment - from a half dozen 1-5ml columns to a 96-well filter plate > on a suction or centrifugal separator. > > Artem > > P.S. since you ask, depending on the brand of GSH resin it also can > survive a few regeneration cycles -- but again depending on various > factors (mostly on what kind of lysates you're loading) the quality > and quantity of eluted protein(s) will inevitably decline with use. > > On Thu, Jul 22, 2010 at 9:05 PM, Matthew Bratkowski <[log in to unmask]> wrote: >> Hi. >> >> We have been using His-Select Resin from Sigma in our lab for a number of >> years now. When we first bought the resin, I usually got much better purity >> of His tagged proteins compared with regular Ni-NTA resin. However, after >> regenerating the resin several times, the level of purity seems to have >> declined. Has anyone else noticed this with His Select? In general, could >> someone suggest the typical "lifespan" of His Select or Ni2+ resin in >> general? What about Glutathione resin? >> >> I was also wondering if anyone had experience using cobalt resin? What is >> the binding capacity of cobalt compared to nickel, and is the selectivity >> any better than either His-Select or regular Ni-NTA? Also, is it possible >> to just strip nickel off of the resin and then recharge it with cobalt? >> >> Thanks, >> Matt >> >