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 wrote: > Thank you all for your suggestions! > > On Mon, Feb 6, 2017 at 5:53 AM, Artem Evdokimov > 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 >> "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 > > 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, dgho...@gmail.com >>> Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) >>> Lab URL: http://www.cdfd.org.in/labpages/computational_functional_gen >>> omics.html >>> >>> >>> >>> - Original Message - >>> From: Alex Lee >>> To: 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.]
Re: [ccp4bb] How to determine the concentration of biotinylated peptide?
Thank you all for your suggestions! On Mon, Feb 6, 2017 at 5:53 AM, Artem Evdokimov 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 > "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 > 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, 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 >> To: 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. >> > >
Re: [ccp4bb] How to determine the concentration of biotinylated peptide?
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 "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 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, 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 > To: 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. >
Re: [ccp4bb] How to determine the concentration of biotinylated peptide?
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, 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 To: 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.
Re: [ccp4bb] How to determine the concentration of biotinylated peptide?
Hi Alex, you can measure the absorbance at 214-220 nm, which is where the peptide bonds absorb, but you should know/calculate/predict the extinction coefficient of your peptide at that wavelength. Furthermore, you might try BCA assay which is colorimetric as the bradford but the reaction involves the peptide bonds (it has low sensitivity but an increase in temperature during the assay might help). Densitometry would be another way but but is give you only a rough idea: I would not recommend to do it. Finally, if you have the possibility to do it, the aminoacid analysis represents the golden standard analysis for a precise quantitation of peptides: this method involves a hydrolysis step, a separation by HPLC, detection. Cheers, Mirella Sent from my iPhone > On 5 Feb 2017, at 22:33, Alex Lee wrote: > > 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. > >
[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.