Peter, Yes, in our hands it can eluted completely. Usually 20 ml of elution buffer per 2 ml of GS4B resin (GE healthcare, fast flow) is enough . We use 30 ml open-columns for purification - each column has 2 ml of resin.
Vitali On Wed, Aug 29, 2012 at 3:02 PM, <hsuu...@u.washington.edu> wrote: > Hi Vitali, > > I usually prep my GST elution buffers fresh, and make sure to pH it after > dissolving the powder. So I don't think that's the problem, but good to > know that you can use as low as 5mM GSH. Do you know if you knock off most > of the protein from the resin at that concentration of GSH? > > Thanks, > Peter > > > On Wed, 29 Aug 2012, Vitali Stanevich wrote: > > Peter, >> >> Such high concentration of GSH may change the pH according to our >> experience. We usually use 50 mM Tris pH=8.0, 5 mM GSH, 3 mM DTT - so that >> you can load the sample on ion-exchange column after elution. If your >> protein is not stable without NaCl - you should add it also. >> >> Vitali >> >> On Wed, Aug 29, 2012 at 12:42 PM, Peter Hsu <hsuu...@u.washington.edu> >> wrote: >> I don't think so since I purify in the presence of reducing agents >> (DTT/BME) and I got activity out of the prep that was released by on column >> cleavage. On the other hand, I don't usually add those >> fresh each time I use the buffers so it's entirely possible the >> reducing agents are nowhere near the initial reduced form they were in >> initially. >> >> On Wed, 29 Aug 2012, Antony Oliver wrote: >> >> GSH will reduce your protein quite nicely - is your enzyme >> activity redox >> sensitive? >> >> --- >> Dr Antony W Oliver >> >> Senior Research Fellow >> CR-UK DNA Repair Enzymes Group >> Genome Damage and Stability Centre >> Science Park Road >> University of Sussex >> Falmer, Brighton, BN1 9RQ >> >> email: antony.oli...@sussex.ac.uk >> tel (office): +44 (0)1273 678349 >> tel (lab): +44 (0)1273 677512 >> >> >> >> >> >> >> On 8/29/12 5:56 PM, "Peter Hsu" <hsuu...@u.washington.edu> >> wrote: >> >> Hi all, >> >> I've been purifying my protein off a GST column and >> have noticed a >> massive difference in activity of my protein between a >> prep that was >> freed from the column via on column cleavage, and a >> prep that was eluted >> (20mM GSH) and then cleaved and further purified. I'm >> suspecting that the >> glutathione is somehow modifying/inhibiting my protein >> in some way, >> despite having removed the glutathione from the buffer >> via dialysis/ion >> exchange. I don't see anything out of the ordinary in >> my electron density >> that would suggest that glutathione has affected my >> protein in some way, >> but the huge difference seen in my activity assay >> suggests otherwise. >> >> My question is, has anyone else seen an effect from >> glutathione affecting >> their protein in some way? My second question is, >> what's the minimum >> amount of glutathione necessary to elute your protein >> from a column? >> >> Sorry for the off topic question and thanks for any >> responses, >> >> Peter >> >> >> >> >> >> >> >
