Re: [ccp4bb] Off-topic: Duet vectors with C-terminal GST tag
Hi Jacob, Thanks for your message! I was thinking about that too, but I wasn't sure if it was something that just sounded good in theory.. I guess the two different vectors should have different antibiotic resistance to select for the 'co-transformation.' Thanks so much! Ming From: Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, August 27, 2012 9:31 AM To: Lye, Ming Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] Off-topic: Duet vectors with C-terminal GST tag How about just co-transfecting with two different plasmids which have different selection markers? I've done it a lot, and it seems to work fine... JPK On Fri, Aug 24, 2012 at 10:39 PM, Lye, Ming ming_...@hms.harvard.edumailto:ming_...@hms.harvard.edu wrote: Dear CCP4bb, We would like to co-express proteins under Se-Met conditions for de-novo phasing of a complex. One of the proteins expresses much better with a GST-tag compared with a his-tag. As its binding site is at its N-terminus, we are hoping to co-express it as a C-terminal GST tag recombinant. So far however, we haven't found any commercially available duet vectors with such a tag. We would truly appreciate if anyone knows of the availability of such vectors, or has any suggestions on similar vectors. Thanks so much! Ming -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu ***
[ccp4bb] Off-topic: Duet vectors with C-terminal GST tag
Dear CCP4bb, We would like to co-express proteins under Se-Met conditions for de-novo phasing of a complex. One of the proteins expresses much better with a GST-tag compared with a his-tag. As its binding site is at its N-terminus, we are hoping to co-express it as a C-terminal GST tag recombinant. So far however, we haven't found any commercially available duet vectors with such a tag. We would truly appreciate if anyone knows of the availability of such vectors, or has any suggestions on similar vectors. Thanks so much! Ming
[ccp4bb] Off-topic: Native gel electrophoresis of basic proteins
Dear CCP4 community, Sorry for the off-topic subject, but I would really appreciate some suggestions and/or protocols relating to native gel electrophoresis of basic proteins. I have used a general acidic PAGE protocol for my protein, which has a PI of 9.5. Briefly, the protein was loaded onto a native gel (I have tried both the pre-made Biorad gels (7.5% and a gradient gel: 4-15%) and freshly prepared Tris native gels adjusted to pH 6.8) and run in a 1X acetic acid/b-alanine pH 4.5 running buffer. The electrodes were reversed and the gel run on ice for ~ 2hrs at 100V. In all cases, the native protein was unable to enter the gel. Some protein samples incubated with heavy atoms were able to enter the gel (possibly indicating binding) but these samples too had problems entering the gel as the bands were at or just a little bit below the edge of the well. Any suggestions and comments would be most welcome! Thank you so much in advance for your help, Sincerely, Ming Lye
