For that purpose we’ve developed this expression system that may or may not be useful to your specific problem: http://www.ncbi.nlm.nih.gov/pubmed/23996492 J Mol Recognit.<http://www.ncbi.nlm.nih.gov/pubmed/23996492#> 2013 Oct;26(10):496-500. doi: 10.1002/jmr.2292. Development of a multifunctional tool for drug screening against plasmodial protein-protein interactions via surface plasmon resonance. Boucher LE<http://www.ncbi.nlm.nih.gov/pubmed?term=Boucher%20LE%5BAuthor%5D&cauthor=true&cauthor_uid=23996492>1, Bosch J<http://www.ncbi.nlm.nih.gov/pubmed?term=Bosch%20J%5BAuthor%5D&cauthor=true&cauthor_uid=23996492>.
Sorry, for the self advertisement (at least it’s not attached), but perhaps it is a useful tool for others as well. Jürgen ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742<tel:%2B1-410-614-4742> Lab: +1-410-614-4894<tel:%2B1-410-614-4894> Fax: +1-410-955-2926<tel:%2B1-410-955-2926> http://lupo.jhsph.edu On Mar 5, 2014, at 8:15 PM, Mo Wong <mowon...@gmail.com<mailto:mowon...@gmail.com>> wrote: Many thanks to all the people who contacted me. I actually went rogue and decided to initially reconstitute in 10mM glycine, pH 9.5 to 2 mg/mL, sonicate for a few seconds and then immediately add an equal volume of more concentrated neutral pH buffer to minimize any possibility of alkaline hydrolysis. The peptide solubilized nicely, and the SPR sensorgrams are much more promising. My only concern is if glycine might interfere with peptide binding (I suspect not, but please let me know if you think otherwise) - a control doesn't suggest glycine likes to stick to the protein surface. A few people pointed me to links on solubilizing lyophilized peptides; however, the one I had previously been consulting is, in my opinion, the most thorough: http://www.bachem.com/service-support/faq/handling-of-peptides/ Thanks again. On Wed, Mar 5, 2014 at 6:52 AM, Toufic El Arnaout <elarn...@tcd.ie<mailto:elarn...@tcd.ie>> wrote: Hello Mo Wong, Some points below are good, but don't underestimate custom peptides sometimes... can be much harder/expensive than recombinant proteins. If you ordered the peptides it is good to know how they synthesized them, and how the elution profiles during purification (RP HPLC..) looked like, particularly that they are very hydrophobic. Did they use formic acid, or maybe they already dissolved them in DMSO.. Did they verify the product/bonds with MALDI-TOF for example and NMR? Furthermore, you can try 0.1-0.5 % Triton X-100. My vote would be for a final concentration of 5-25 uM (containing up to 5 % solvent), from a stock of 50-100 % solvent at 1 mM.. Btw I forgot.. for SPR (depends on the system, and especially for a 12mer), some detergents and even a minor DMSO/ethanol can affect the measurements drastically. Best wishes toufic el arnaout On Wed, Mar 5, 2014 at 12:19 AM, Mo Wong <mowon...@gmail.com<mailto:mowon...@gmail.com>> wrote: Hi all, Slightly off topic - but I'm having trouble solubilizing some peptides for SPR and hoped someone on the BB might have some other suggestions. The peptides are intra-chain S-S cross-linked 12mers with pIs of ~3. 10 residues are hydrophobic and 2 are acidic. Peptides have been tested with and without N- and C-terminal modifications (amidation/acetylation). I have tried: ddH2O raising (and lowering) pH (tested up to 8.5) with different buffers Including DMF as a co-solvent (I'm avoiding DMSO because of a methionine present in the peptide) - peptide still visibly precipitates out at 100uM in 5% DMF (also a 1mM stock in 50% DMF shows significant amounts of ppt) Adding a trace amount of detergent (0.005% Tween 20) I'm guessing I could try other co-solvents such as ethanol or initially solubilizing peptide in dilute NaOH before bringing the pH down with addition of a buffer (though I'm concerned about alkaline hydrolysis). Anyway, I'd rather have some insight from people before I waste any further peptide. Thanks for any suggestions.
