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Hi Ashok, I second Bostjan's questions and wanted to emphasize one small point. Check the void volume for your gel filtration column, on the system you ran the other experiments on. Based on the elution volume you mention, for typical performance of that size of Superdex 200pg column, your material is probably a discrete oligomer, larger than a dimer. That isn't necessarily a problem for crystallography, and may even suggest some interesting biochemistry or a particularly non-globular shape. Given the apparent size, it also probably isn't simply an unfolded monomer (which also run larger than the expected monomer size). So that is actually rather promising. My minor concern is that you are close enough to the predicted void volume that your material might actually be eluting at the void volume, depending on the condition of the column and how your system is plumbed. Have you run the void volume calibration? Typically this is done with something like blue dextran 2000. If you material is definitely not running at the void, further biophysical characterization is probably warranted. However, if the material is eluting in the void, you may be dealing with a soluble aggregate instead of a discrete oligomer, which is a poor indicator for crystallography. If in the void, you could try Superose 6, which resolves large species than SD200. Good luck! Shae Padrick > On Feb 24, 2016, at 3:22 AM, 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 ________________________________ UT Southwestern Medical Center The future of medicine, today.