Hi Trudy,
There is another way (I have never done it).
Attach at the end of the synthetic peptide a benzoyl benzoic acid group
by treating the reagent as the terminal residue during normal synthesis.
This blocks the amino terminus.
Dissolve 5 mg KLH in 0.5 ml of 0.01 M sodium phosphate buffer, pH 7.5.
Add 2mg of synthetic peptide containing the benzoyl benzoate adduct.
Place in a 1cm quartz cuvette and irradiate with 366 nm light for 3 hr
at a distance of 0.5 cm.
Use immediately directly for immunization.
Anne
>Hi All,
>I would like to follow up on MAP vs other methods for peptides for antibody
>production. I have been asked to make peptides for monoclonal antibody
>production that have internal Cys residues. I usually add a Cys to the N-
>terminus and couple to maleiminde activated KLH after purification. The user
>thinks the internal Cys residue is critical and so it can't be blocked with
>ACM etc. The sequence is from a Cys rich region so it doesn't help to move
>up or down the sequence.
>
>My suggestion was to use a 4 branched MAP core, try and purify by HPLC and
>send it for mass spec. The sequences are 9 AA long.
>
>Does anyone have a better idea?
>Thanks in advance for your help.
>
>Trudy Holyst
>Blood Research Institute
>Peptide Core Laboratory
>Blood Center of SE WI
>Milwaukee WI 53201
>
>
>-----Original Message-----
>From: fperini@unmc.edu [mailto:fperini@unmc.edu]
>Sent: Monday, May 15, 2000 11:35 AM
>To: Recipients of ABRF List
>Cc: Recipients of ABRF List
>Subject: Re: quality control MAP peptide for antibodies
>
>
>
>First of all,pay heed to Nadine Ritter's questions.It is pretty much what I
>do,I tell the
>investigators the facts,and let them deide what is proper.I have been doing
>amino acid
>analysis,which is the most straightforward method of quality control.Mass
>spec analysis of
>MAPs has been done successfully before,but if your amino acid analysis is
>bad,the Mass spec
>and sequencing,as well will be worse.The HPLC profile is very indicative of
>what you have.
>Steric problem occur after five or six residues,and the more branches the
>worse is the
>problem.I recommend to try four and eight branch synthesis,and see how they
>look.In most
>cases eight-branch MAPS are not tried anymore.Longer reaction times,rather
>than repeated
>couplings are just as effective.Capping is not so simple in Fmoc
>chemistry,and you would
>have to see if it would help.Change of solvent(s) during the synthesis,to
>keep the chain
>from bending backward should help.And,finally,I tried HPLC with a
>denaturant(urea),and
>some separations improved.I would do away with questionable MAPs any
>time,in favor of
>conjugation of a pure peptide conjugated to a carrier protein.
>
>
>
>
>"alex bell" <ehjb@musica.mcgill.ca>@aecom.yu.edu> on 05/12/2000 09:06:00 PM
>
>Sent by: Association of Biomolecular Resource Facilities
> <abrf-request@aecom.yu.edu>
>
>
>To: Recipients of ABRF List <abrf@aecom.yu.edu>
>cc:
>
>Subject: quality control MAP peptide for antibodies
>
>
>
>Dear All,
>
>What are some of the quality controls that are employed for MAP synthetic
>peptides for raising antibodies?
>
>How much branching is recommended?
>
>Are missed amino acids (deletion sequences) at a low frequency a concern?
>Triple coupling synthesis procedure employed but no capping.
>
>Mass spect analysis? Amino acid analysis? Sequence (Edman or MS/MS)
>analysis?
>
>Thanks in advance,
> alex
>
>
>
>Alexander Bell
>McGill University
>Anatomy & Cell Biology
>Strathcona Bldg.
>3640 University
>Montreal, Qc. H3A 2B2
>
>phone (514) 398-1393
>fax (514) 398-5047
>email ehjb@musica.mcgill.ca
.-. .-. .-. .-. .-. .-. .-. .-. .-.
`-' `-' `-' `-' `-' `-' `-' `-' `-'
Anne Stanley
Macro Core Facility
PSU Hershey PA
Tel (717) 531 6087
Fax (717) 531 7266
http://www.collmed.psu.edu/core
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