Hi Lisa, Speed is definitely a big factor here. With a protein I work with I can get large crystals in myriad conditions that only diffract to about 4-5 Ang. What I ended up doing was taking these crystals and seeding entire screens. I found that not only would crystals appear sooner but it revealed novel crystallization conditions. These seeded crystals would appear within minutes as Preben described and diffracted to better than 2 Ang. Another thought would be to try limited proteolysis to see if you can identify a more stable construct.
-John -- John Domsic Postdoctoral Fellow Gene Expression and Regulation Program The Wistar Institute Philadelphia, PA 19104 On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth <j.p.mo...@ncmm.uio.no>wrote: > Dear Lisa > It is not uncommon to see breakdown products when you run crystals on > gel. Espesially if they are older crystals, sometimes you even see higher > molecular bands, these are probably due to intra molecular cross links > formed over time. > If you are worried about stability, try to increase the crystallization > speed, we have one example where we see a clear difference in both crystal > quality and even space group depending on when we fish the crystals. The > crystals appear within 5 min, the best quality data sets come from > crystals we fish after only 30-60 min. > You may also have a little protease contamination of course, to prevent > this add protease inhibitor, or DTT, or EDTA to you protein before you set > it up. > cheers Preben > > > On 1/16/13 12:14 PM, LISA wrote: > >> Hi All, >> I have an 36KD protein which can be crystallize in two days. Most of the >> crystals are very big. But all cystals have poor resolution,lower than 3.8 >> A. I picked some crystals, washed them in the mother solution and then run >> SDS-PAGE. It is surprised to find that different cystals have different >> components. Some crystals have several samll bands below the band of the >> protein. And in some crysals the bigger size band (as the construct should >> be) almost disappared and have smear. Does the protein was degradated in >> the crystals? Did someone met the similar problem as I? Thanks >> >> All the best >> lisa >> > > -- > J. Preben Morth, Ph.D > Group Leader > Membrane Transport Group > Nordic EMBL Partnership > Centre for Molecular Medicine Norway (NCMM) > University of Oslo > P.O.Box 1137 Blindern > 0318 Oslo, Norway > > Email: j.p.mo...@ncmm.uio.no > Tel: +47 2284 0794 > > http://www.jpmorth.dk >
