Re: [ccp4bb] How to determine the concentration of biotinylated peptide?

[Nicholas Larsen]

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?

[Artem Evdokimov]

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?

[Debasish Kumar Ghosh]

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?

[Vivoli, Mirella]

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?

[Alex Lee]

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.