[ccp4bb] {Spam?} quick purification?
Hi All A little off topic, but I am thinking about expressing and purifying 40+ mutants, for an assay, at maybe 1mg total protein each. I'd like the purification to be quick and easy (ie. one step) but cleaner than just a 6his-tag purification. Currently, my options are either GST, a longer his tag or a strep-tag. Any thoughts on the comparison between GST and strep and Ni purifications would be much appreciated. OR are there any other (better) high affinity purifications, I've missed, excluding expense Ab resins. Thanks, Mark Collins
[ccp4bb] Subject: off topic, purification from insect media and TFF/CFF
Hey, I have a GST fusion protein secreted into express5 (with hint of serum), it does not bind beads. Changing pH with 4xPBS limits ppt and enables binding but in a scale up I think it makes sense to use a TFF/CFF setup for concentration and diafiltration. Does anybody have advice about purifying proteins from insect media? Is TFF the right choice? And any recommendations for TFF system/brand will be appreciated. Cheers, Mark
Re: [ccp4bb] OT: VectorNTI alternatives - 4 Mac?
Hi Anybody have suggestions for Mac OsX alternatives? Thanks in advance, Mark
Re: [ccp4bb] Caution - 120 Hz LCDs: Not CRT killers yet...
Hi Has anyone tried using video glasses such as MyVu as stereo glasses? www.myvu.com Seems like it could be a cheap (~$100/set) alternative to montitors + other hardware. Resolution maybe an issue. Just a thought, Mark
Re: [ccp4bb] Crystal growing in one direction...need suggestions
Hi I also like to try the Hampton additive kit or the easier version; 90% current condition (at 1.1x) and 10% of any screen you can lay your hands on (ie hampton 1 2). Sitting verse hanging drops can change crystal size. Also in situ limited proteolysis, crazy but it works see for more info and references. Nature Methods - 4, 1019 - 1021 (2007) In situ proteolysis for protein crystallization and structure determination Mark On Tue, 14 Oct 2008, shivesh kumar wrote: Dear all, I have crystallized a protein in MPD which is thin and growing in one direction only. I have tried ethonal, DMSO, Proline, Glucose, Urea, Sucrose, Detergent kit from Hampton and lot more alongwith temperature variation. Need suggestions regarding improvement of xtal quality, THICKNESS. Thanx in advance. Shivesh
Re: [ccp4bb] Expression vector with NdeI-ClaI sites
Or do the new school cloning, SLIC (Sequence Ligation Independent Cloning) and subclone into any position in a vector. This method uses a PCR product and vector, the PCR primers have 20-40bp overlap with a region in the vector. Mix cut and purified vector with PCR product. Digest with T4 polymerase, quench, and transform. When I do the PCR in the morning I have clonies the next day. REF: Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC Mamie Z Li Stephen J Elledge Nat Meth V4(3) pp 251 Mark -- Mark Collins Columbia University Biochemistry Molecular Biophysics Black Building 259/201 Office/Lab 212 305 1951 (work) [EMAIL PROTECTED]
