I recently ran mass spec analysis on some crystals that I had obtained from an optimization screen. I was looking for modifications in the protein. In order to get enough signal, I had to harvest and dissolve about 8 crystals roughly 0.3 x 0.15 x 0.15mm into the MS loading buffer in order to get a strong enough signal for the experiment. Alas, I did not find the modification I had been looking for, but I am not surprised that one single crystal, or even a small cluster of them did not show a band on a coomassie-stained gel. Do you A) have a MS handy, and B) can you sacrifice ~8 crystals to get enough S/N for a good experiment?
Good luck! From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Jim Pflugrath Sent: Tuesday, November 02, 2010 10:19 AM To: [email protected] Subject: Re: [ccp4bb] Crystal gel band It reads like you need to run a lane or two with a positive control of some kind. Can you grow lysozyme, glucose isomerase, hemoglobin or other crystals of a protein around the same expected molecular weight and try run on the gel lanes with about the same amount of crystalline volume as your putative protein crystals? ________________________________ From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of xaravich ivan Sent: Monday, November 01, 2010 9:51 PM To: [email protected] Subject: [ccp4bb] Crystal gel band Hi everyone, I have grown some crystals after micro-seeding starting from thin-small needles from needle-clusters. These crystals are larger in size than the needles but are comparable to the shape and don't look like salt crystals. But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a home source,handy and would like to send these to the synchrotron. Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, the amount of protein is < 1uG? Has anyone experienced such a thing (no band in gel, but crystal diffracts)? It would be nice if I get observations/suggestions. ivan -------------------------------------------------------------------------- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
