Re: [ccp4bb] alternatives to IZIT
Whatever dye(s) you use, be sure to run some positive and negative controls to see how the dye really works. Jim On Sat, 14 Jun 2008, Mark Del Campo wrote: Before I place an order for some Izit, are there some other dyes I can use to check if I've got a protein crystal? Thanks, Mark
Re: [ccp4bb] alternatives to IZIT
Several people have asked about the concentrations we use for various dyes. In the case of fluorescein, we started with 45mg/ml as a stock solution. This is quite arbitrary and we often found that our initial stock solutions needed to be diluted 1:10 or 1:100 before use. It is more art than science. We also did most of our work actually growing crystals in the dye rather than using as we would IZIT. So the short answer is: you'll have to experiment. You want to inject just enough dye into the crystallization drop to give a mild coloration. It is probably wise to dilute your stock with well solution, then inject the smallest volume possible so as not to dissolve your crystal. Then, over time the drop becomes clear as the protein crystal absorbs and concentrates all the dye. If you put too much dye in the drop, the drop is not as clear and it is harder to distinguish crystals from colored precipitate. We have also seen crystals of dye form under higher concentrations. I am not aware of any salt crystals that are known to take up dye. It is presumed that the dye is absorbed by the protein crystals because of solvent channels (of which salts have none). We observed even very low solvent content crystals to take up dye. A negative result, however, does not rule out protein. We did see a case in which dye prefers to remain in solution rather than in crystal. I believe this was high PEG conc. Under these typical IZIT-like staining conditions, we have not observed any anomalous signal due to heavy atoms. This is presumably because the concentration of dye in crystal is too low. At least, that argues against any dramatic changes in crystal quality as a result of dye. Richard On Jun 16, 2008, at 11:18 AM, Jim Pflugrath wrote: Whatever dye(s) you use, be sure to run some positive and negative controls to see how the dye really works. Jim On Sat, 14 Jun 2008, Mark Del Campo wrote: Before I place an order for some Izit, are there some other dyes I can use to check if I've got a protein crystal? Thanks, Mark
Re: [ccp4bb] alternatives to IZIT
Best of all, run it on a gel. I am pretty sure it is just methylene blue. You can also see what kind of diffraction pattern the crystal has, or just squish it and see if it crumbles. On Jun 14, 2008, at 1:51 PM, Mark Del Campo wrote: Before I place an order for some Izit, are there some other dyes I can use to check if I've got a protein crystal? Thanks, Mark
Re: [ccp4bb] alternatives to IZIT
Mercurochrome (mebromin) is both a derivative AND a stain (tested, true). It's just like having a pie and eating it at the same time. Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Richard Gillilan Sent: Sunday, June 15, 2008 9:36 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] alternatives to IZIT We've tested eight different dyes carefully on three different proteins and eventually planned to test a much larger number (I''ve assembled a large collection of dyes of every color and type). So far everything has worked! (rhodamine, bromophenol blue, and fluorescein are my favorites because they are fluorophores). You could probably pick almost anything out of HJ Conn's book Biological Stains. I believe there was some trouble with mixes containing high PEG conc, but I don't recall the details at the moment. You might even consider using a dye with high bromine content (like bromophenol blue) to phase the structure, but that would require much higher concentrations than are typically necessary in ISIT-like experiments. We did see some evidence of change in crystal morphology when the proteins where grown with the dyes as opposed to soaked. Not sure how significant that effect is. Obviously a dye could potentially induce conformational or oligomeric changes in some cases. Richard Gillilan MacCHESS Ithaca, NY On Jun 14, 2008, at 4:51 PM, Mark Del Campo wrote: Before I place an order for some Izit, are there some other dyes I can use to check if I've got a protein crystal? Thanks, Mark
[ccp4bb] alternatives to IZIT
Before I place an order for some Izit, are there some other dyes I can use to check if I've got a protein crystal? Thanks, Mark
Re: [ccp4bb] alternatives to IZIT
I am pretty sure it is just methylene blue. You can also see what kind of diffraction pattern the crystal has, or just squish it and see if it crumbles. On Jun 14, 2008, at 1:51 PM, Mark Del Campo wrote: Before I place an order for some Izit, are there some other dyes I can use to check if I've got a protein crystal? Thanks, Mark
