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Hi Ashok, I am afraid you are working with a protein that elutes in the void volume of S200 column (my first thought: is it an aggregate). Try AUC or native gel to see if it is a real huge aggregate if no aggregation then running on Suprose column will help. I would do this even before thinking about crystallization. Good luck On Wednesday, February 24, 2016, Ashok Patel <[log in to unmask]> wrote: > Dear All, > > I am working on protein 28 KDa, Construct has been made in pET28a vector > with N terminal 6XHist tag, Protein expression has been optimised. > > Protein is purifying nicely on NiNTA resin, as found on SDS-PAGE at > expected mol weight 28-30 kDa. > > Since the protein we want for crystallization and structure determination, > We are applying it on Superdex 200 (HiLoad 16/60, 120 ml column) size > exclusion chromatography, all protein is coming only around 45-50 ml, but > on SDs-PAGE single protein around 30 kDa. > > So it seems oligomerization. > > we tried Size exclusion S200 with or without concentrating the NiNTA > fractions 1mg/ml or around 10 mg/ml, with or without cleaving His tag, in > all cases, protein is showing peak at 45-50 ml. > > our Size exclusion buffer is 50 mM Tris pH 7.5, 500 mM NaCl, 10 % > Glycerol, 1 mM DTT. > > How to proceed? what should we try for getting the monomeric protein.? any > suggestions? can we still try to set crystallization? > > thanks in advance > > with regards > Ashok > IIT Delhi > -- *Rashmi*