Hi Marta, 1-- There is always a possibility of forming detergent/lipid (cholesterol in your case) -containing crystals combined with other small molecule(s) (organic or not). Although you did not specify the width of the oscillation used to collect the frame displayed in your mail (was it 45 degrees or 0.5 degrees?) I see signs of a lattice at least on the vertical direction (3 rows can be distinguished it seems), can you derive a "cell-dimension" along that direction? 2- If you have access to a fluorescence-coupled light microscope (like a a PRS-1000 Korima instrument) maybe you could see if your crystals glow (provided your proteins contain tryptophans). Crystals from drop 1 seem better candidates (they look 'thicker') for a fluorescence scan, the ones from drop 2 are really thin and curvy, so it might be difficult to see in that case. This could help you characterizing and ruling things out more easily. 3- You mentioned that you analyzed the content of washed/dissolved crystals by silver-stained SDS PAGE. Forgive me for asking if you also analyzed the last wash step on this sivler-stained SDS PAGE gel to rule out that the protein signal you observed was not cross-contamination by a carry-over effect.
Good luck. Good luck On Wed, Oct 24, 2018 at 1:31 PM marta borowska <marta.t.borow...@gmail.com> wrote: > Dear all, > > I have grown crystals of a membrane protein complex, that I initially > verified on SDS-PAGE. These crystals grew only if membrane protein > component is there. The condition is polypropylene glycol 400, > cryoprotected with ethylene glycol, sample buffer has 150mM NaCl on top of > detergent with cholesterol. These long thin needles are quite sturdy and > either didn't diffract or diffracted like small molecule (I cannot exclude > the possibility of contamination stuck around crystal). After the > synchrotron trip harvested crystals were washed (most did not dissolve in > Urea or NaOH!), dissolved in 10% mild detergent and still showed protein > from 4-6 crystals when analyzed on Silver Stain SDS-PAGE. > > I would appreciate your input on whether some of you encountered similar > patterns and if you think I should proceed with more optimization on these > crystals. > > Thank you, > Marta > > > -- > > Marta T. Borowska > > Graduate Student > > Adams Lab > > Department of Biochemistry and Molecular Biology > > The University of Chicago > > 929 E. 57th Street > > Chicago, IL 60637 > > Lab: GCIS W229 > > Lab phone: 773-834-0660 > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pegea at mednet.ucla.edu ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
