Re: [ccp4bb] Additional band on gel due to his-tag: any references?

Hi Sebastian, Under the assumption that the SDS in your assay does not completely unfold the protein during electrophoresis (chemical impurity can be excluded because of MS experiments, right?), how about adding some urea additionally to the SDS-PAGE (or changing SDS concentration)? GL Jan

[ccp4bb] AW: [ccp4bb] Heme Proteins

Hi Hari, These vectors are indeed not commercial ones. Unfortunately, I do not have any of them any more. If I were you, I would directly ask the group where they were originally made - not sure if they are always reading the ccp4 bulletin board though. Anyways, here are some more things about

[ccp4bb] AW: [ccp4bb] Heme Proteins

Hary, Delta-aminolevulinic acid is the classical heme (heme b) precursor for your experiments. As far as I know, the synthesis of porphyrin goes very quickly in E. coli, since I tried it once with a bacterial P450 and got a fat overexpression band after ~2hrs in the lysate. Also the photometri

[ccp4bb] AW: [ccp4bb] freezing crystals grown in isopropanol condition

Hi Chris, Isopropanol can act as a cryoprotectant. Can't say whether 25% are enough. It dependes also if there are other ingredients that can help to get cryo conditions (e.g. salts, PEGs, glycerol). Maybe you want to check the mother liquor if there will be any ice ring formation after freezin

[ccp4bb] about comparing Rwork and Rfree factors of different refinement trials

Dear all, I do have a question about comparing Rfree and Rwork factors of different refinement trials whereas I always started with the same pdb file and structure factors (phasing by MR). Means I had a protein structure which was (not just by me) refined several times in different ways also wi

[ccp4bb] AW: [ccp4bb] off topic: multiple structural sequence alignment

Hi Ronnie, the (a) Mammoth tool might also work (but I just tried 6 structures or so when I used it a couple years ago - not 20). Jan --- Ronnie Berntsson schrieb am Di, 12.1.2010: Von: Ronnie Berntsson Betreff: [ccp4bb] off topic: multiple structural sequence alignment An: CCP4BB@JISCMA

[ccp4bb] AW: [ccp4bb] FW: [ccp4]: TDS upon flashcooling

Did you also try a cryo salt (e.g. Li+)? In the best case the xtals might even grow in there. GL Jan --- Natalie Zhao schrieb am Di, 15.12.2009: Von: Natalie Zhao Betreff: [ccp4bb] FW: [ccp4]: TDS upon flashcooling An: CCP4BB@JISCMAIL.AC.UK Datum: Dienstag, 15. Dezember 2009, 13:20 -Orig

[ccp4bb] AW: [ccp4bb] off-topic: crystal optimization without buffer

Hi Katja, it makes perfect sense to add a buffer to your assay. Of course for the beginning something which buffers well around pH 7 like HEPES, BTP, etc. would be appropriate. If your purification protocol is optimized I would not change the conditions of the purification buffer. But this migh

[ccp4bb] AW: [ccp4bb] Refolding of Denatured Protein

Urea can modify your protein. Maybe you should also try guanidine HCl. Also, did you try any detergents? Good luck! Jan --- Sanjiv Kumar schrieb am Di, 5.5.2009: Von: Sanjiv Kumar Betreff: [ccp4bb] Refolding of Denatured Protein An: CCP4BB@JISCMAIL.AC.UK Datum: Dienstag, 5. Mai 2009, 13:27 I a

[ccp4bb] Xtals: protein or something else?

Hello everybody, I am dealing with the standard problem that I have (spherical) crystals and I want to completely make sure that it is nothing else than protein. Since the amount of protein I am working with is very limitated, I want to get the maximum of information out of the few xtals I have.

[ccp4bb] AW: [ccp4bb] hi / cloning stuff

Hi, I think your question is quite reasonable no matter what others say about it. There were even people asking about how to make buffers and then a big discussion about the HH equation arose. And we should not forget that modern crystallization starts with cloning ;-) Anyway, you should have gi

Re: [ccp4bb] into the looking glass

Hey guys, what day is today? I think this was very funny... James Holton <[EMAIL PROTECTED]> schrieb: Dear CCP4BB, I think it prudent at this point for me to announce what could be a very old, but serious error in the fundamental mathematics of crystallography. To be brief, I have unco

[ccp4bb] Color of heme containing Xtals

Hello everybody, I wonder if anybody has experience with heme (or to be more precise: heme b) containing proteins which Xtals do not look red under the microscope. How might the technique for crystallization (e.g. sitting drop, hanging drop) influence the intensity of the color? Many thanks!

[ccp4bb] Thermal melt and heme in the protein

Dear all, To optimize my crystallization conditions I would like to do thermal melts but unfortunately, there is heme in the molecule which messed up my experiments. The dye I used is colored orange ("SYPRO orange"). Has anybody an idea if it is possible to get serious results with an appropria