Xiao, You could be the victim of the dreaded PEG cross-linking effect. One of the unfortunate by-products of keeping PEG stock solutions in water is that they will form peroxides and aldehydes. They will slowly cross-link the surface of some crystals. However, it is dependent on the nature of your protein's composition of surface residues, so not every protein crystal does this.
I had one case where PEG4000 grown crystals would be resistant to dissolving and would easily bend (just like Herman related); the thinner rods would spring back straight. After placing the crystals into buffer known to dissolve them, I poked the crystals hard and the insides squeezed out like toothpaste, leaving an empty sack behind. The bottom-line is that fresh crystals diffracted better than old crystals because of this cross-linking. Suggestions: 1) make your PEG stocks up fresh or store them in the freezer as aliquots. 2) Remove oxidized PEGs from your stocks: see Ray et al. Biochemistry 1991, 30, 6866-6875 and Jurnak, J. Cryst. Growth, 76, 577-582, 1986. 3) Check to see if freshly grown crystals behave better. Best of luck, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com **************************************************************** On Sep 25, 2014, at 7:53 PM, Xiao Xiao <victor41...@gmail.com> wrote: > Hi everyone, > > Sorry for an off-topic question. > > I got a problem with crystal dissolving. Basically I got crystals of my > protein in various conditions, most conditions contain PEGs but different > salts. These crystals has very similar shape, so it should not be salt. > > Now I am trying to dissolve the crystal to make sure it is my protein, by > SDS-PAGE and N-term sequencing. I washed the crystals in its original > crystallization buffer few times then transfered them into regular buffer > (500mM NaCl, 50mM HEPES 7.5) with or without 10mM DTT, however the crystal > didn't dissolve. > > I then tried to heat it then add SDS loading buffer to run a gel, I did see > very small amount of protein on the gel, at the correct position, but it's > not enough for N-term sequencing. > > Is it normal for a protein crystal? And does anyone have any suggestion for > dissolving such crystals? >
