very good point! you need lots of protein and it has to be pure (meaning also minimal buffer, salts and stuff) but it worked pretty well in our hands (when we were trying to measure the extinction coefficient of phytochrome). incidentally, I think that the notion of quantitative amino acid analysis being the gold standard is wrong. we had a sample analysed by different labs in different parts of the world – and the results varied by about 50%! maybe we were just unlucky, but maybe we won't be the only ones.... best jon
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Nicholas Larsen Gesendet: Montag, 6. Februar 2017 19:01 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] How to determine the concentration of biotinylated peptide? These suggestions are all possible, but why not simply lyophilize it into a tared tube and weigh it out? On Mon, Feb 6, 2017 at 12:28 PM, Alex Lee <alexlee198...@gmail.com<mailto:alexlee198...@gmail.com>> wrote: Thank you all for your suggestions! On Mon, Feb 6, 2017 at 5:53 AM, Artem Evdokimov <artem.evdoki...@gmail.com<mailto:artem.evdoki...@gmail.com>> wrote: Hi, In addition to HABA dye assay (which will work great but will also be fooled by any biotin that is not conjugated) you can do: * quantitative MS * TLC * HPLC * elemental analysis * https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614710/ biotin catalysis of the N3- + I3- reaction (also fooled by free biotin of course) * UV (but beware, biotin only absorbs strongly below 240nm so you're not super well off there Artem www.harkerbio.com<http://www.harkerbio.com> "all of our Biotin comes only from free-range gummy vitamin bears..." - Cosmic Cats approve of this message On Mon, Feb 6, 2017 at 2:03 AM, Debasish Kumar Ghosh <dkgh...@cdfd.org.in<mailto:dkgh...@cdfd.org.in>> wrote: Hi Alex, In addition to Mirella's suggestion I would like to make an addition which might be specifically useful for you. Since your peptide has biotin tag, You may use HABA dye assay for the exact quatifiation of biotin (and thus biotinylated peptide). As far I recall, Thermo scientific provide a kit for this assay. The assay is simple and gives accurate results. Best !!! Debasish CSIR- Senior Research Fellow (PhD Scholar) C/o: Dr. Akash Ranjan Computational and Functional Genomics Group Centre for DNA Fingerprinting and Diagnostics Hyderabad, INDIA Email(s): dkgh...@cdfd.org.in<mailto:dkgh...@cdfd.org.in>, dgho...@gmail.com<mailto:dgho...@gmail.com> Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html ----- Original Message ----- From: Alex Lee <alexlee198...@gmail.com<mailto:alexlee198...@gmail.com>> To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Sent: Mon, 06 Feb 2017 03:02:07 +0530 (IST) Subject: [ccp4bb] How to determine the concentration of biotinylated peptide? Dear All, Sorry for the off-topic question, I'd like to do Biacore SPR assay with N-terminal biotinylated peptide as ligand (to Biacore SA chip) and my protein as analyte. I have a question of how to determine the concentration of biotinylated peptide （synthetic peptide), if the peptide has no Tyr and no Trp residue, I guess amino acid analysis may not work because the N-terminal of the peptide is biotinylated. I'd appreciate if anyone share his/her experience on this. [This e-mail message may contain privileged, confidential and/or proprietary information of H3 Biomedicine. If you believe that it has been sent to you in error, please contact the sender immediately and delete the message including any attachments, without copying, using, or distributing any of the information contained therein. This e-mail message should not be interpreted to include a digital or electronic signature that can be used to authenticate an agreement, contract or other legal document, nor to reflect an intention to be bound to any legally-binding agreement or contract.]
