Hi, I do have 10% glycerol in my buffers, and still the constructs come in the void volume. and I have sarkosyl in the lysis buffer. but none in the elution or dialysis buffer. So do I still need detergents .... please suggest. reg. Anita
On Sat, Aug 27, 2011 at 12:13 AM, Pius Padayatti <ppadaya...@gmail.com>wrote: > Answer is simply no. > aggregates are of no value > why would you try it? > you could try adding glycerol to 10 % during the > preparation( all the way from homogenization) itself and try detergents > like c8e4 and series of that. > i know one membrane associated protein need > detergent from the begining itself. > Please do not chop off the membrane part keep it > and chop some of the unstructured cytosolic part if you want. > all the best. > let us know if any of these worked > Padayatti > > On Fri, Aug 26, 2011 at 3:03 AM, anita p <crystals...@gmail.com> wrote: > > Hi All, > > I am working on a protein which has a membrane spanning region and as > > cytosolic domain.I have made various deletion constructs of the protein, > so > > that I can have a crystallizable fragment. There is no homologues > mentioned > > in the pdb for this protein. > > All of these constructs are purified successfully but when concentrated > and > > loaded on a gel filtration column Superdex-200, they elute in the void > > volume. But the proteins donot precipitate out.... !! > > Is it worth while to go ahead for crystallization trials?? > > Any other suggestion is most welcome. > > Thanks > > Anita > > > > > > > > -- > Pius S Padayatti,PhD, > Phone: 216-658-4528 >
