Re: [ccp4bb] protein cleavage

Since you're seeing the MBP band, it sounds as if you're getting some cleavage. If there were no cleave you would only see the fusion and TEV bands on the gel. It may be that your protein is not stable/soluble without the MBP fusion. Different buffer conditions may help if your protein is

Re: [ccp4bb] protein cleavage

...@jhsph.edumailto:jubo...@jhsph.edu wrote: @Cynthia, On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote: Since you're seeing the MBP band, it sounds as if you're getting some cleavage. If there were no cleave you would only see the fusion and TEV bands on the gel. I think that is a wrong assumption

Re: [ccp4bb] [OT]: Purification in ArcticExpress

Incubation with MgCl2/ATP/KCl facilitates the release of the Cpn60. See: R. E. Joseph; A. H. Andreotti, Protein Expr. Purif. 2008, 60, 194–197. Best, Cynthia __ Cynthia Kinsland, Ph. D. Director, Protein Production Facility Cornell University B78 ST Olin

Re: [ccp4bb] His Purification

Another option is to try NEB NiCo21(DE3) cells. http://www.neb.com/nebecomm/products/productC2529.asp I have no relation to NEB beyond being a customer. They've mutated GlmS to eliminate binding to IMAC resins and have added chitin affinity tags to SlyD, ArnA and Can to allow simple post-

Re: [ccp4bb] TEV nucleotude sequence with restriction site

I'm not quite sure what you want, but I have a series of vectors encoding various N-terminal tags and fusions, all followed by a TEV site. They have an MCS standard to many pET vectors. Therefore, they are designed to clone your gene in using the NdeI site at the 5' end (which will,

Re: [ccp4bb] problem in transformation of pqe 30 clone

Using pQE30, any E. coli is an expression host. Because it uses the T5 promoter, you don't need an E. coli strain carrying the T7 RNA polymerase (so, you don't need a DE3 strain). As noted by Artem, you are most likely having a leaky expression problem. However, it is odd that DH5a will

Re: [ccp4bb] OT: VectorNTI alternatives - 4 Mac?

There is GENtle which has a whole slew of tools associated with it. There are versions for several platforms. http://gentle.magnusmanske.de/ If you're used to Vector NTI, it is pretty similar (and open source for the ambitious). _ Cynthia Kinsland, Ph. D. Director

Re: [ccp4bb] Semet in non-auxotrophic strains

PROTECTED] *** Cynthia Kinsland, Ph.D. Cornell University Protein Facility Director 607-255-8844

Re: [ccp4bb] Engineering disulfide bonds

modern man in a post-modern world MacCHESS, Cornell University [EMAIL PROTECTED] Cynthia Kinsland, Ph.D. Cornell University Protein Facility Director 607-255-8844

Re: [ccp4bb] chromatofocusing

? I've seen references to this (although I suspect the resolution won't be as good), but not very good descriptions. thanks, Nat Cynthia Kinsland, Ph.D. Cornell University Protein Facility Director 607-255-8844

Re: [ccp4bb] Off topic: where to order just miniprep columns?

to be perpetually running out of the columns. Thanks Eric -- D. Eric Dollins, Ph.D. C266 LSRC, Research Dr. Duke University Medical Center Durham, NC 27710 (919) 681-1668, [EMAIL PROTECTED] Cynthia Kinsland, Ph.D. Cornell University Protein Facility Director 607-255-8844

Re: [ccp4bb] Protein expression in Minimal media (M9)

. * Cynthia Kinsland, Ph.D. Cornell University Protein Facility Director 607-255-8844

Re: [ccp4bb] protease cleavage sites

to Cognition Wellcome Trust Sanger Institute Hinxton Cambridge CB10 1SA Work Tel: 0044 (0)1223 834244 ext. 7318 Cell No.: 0044 (0)7870 208280 === Cynthia Kinsland, Ph.D. Cornell University Protein Facility Director 607-255-8844

Re: [ccp4bb] human cDNA clones?

opLlcpYxP7uzicyX0B6U4aY= =NH4Q -END PGP SIGNATURE- Cynthia Kinsland, Ph.D. Cornell University Protein Facility Director 607-255-8844