anti-peptide antibody

Jagreet Kaur jagreet at ccmb.ap.nic.in
Sat Jun 10 11:22:13 EST 2000


Hi All
Thanks a lot for the suggestions posted in reply to my query.

Here are the replies thatI got:
From: Jasmina Kurepa <jkurepa at facstaff.wisc.edu>

I was working on a protein which was also a member of a multigene
family, so my only solution were the anti-peptide antibodies. Good news
is that it worked really good.

My basic steps were:

1. Prediction of hydrophylicity based on Hoop and Woods (PNAS USA 78;
3824-3828 from 1981). That was done with the program ANTIGEN which was a
part of the PC/Gene swite (I guess there must be a stand-alone program
somewhere on the web).

2. Prediction of chain flexibility based on Karplus and Schultz
(Naturwissenshaften 72: 212-213 from 1985). That was cone bu the program
FLEXPRO from PC/Gene.

3. Peptide was coupled to the kayhole limpet hemocyanin as in Mumby et
al (PNAS USA 83: 265-269 from 1986).

4. I injected 3 rabbits and only one gave reasonably good antisera - I
had to purify the IgGs and concentrate before it become good antisera.
Parts of my ordeal are in Kurepa et al in Plant Physiology and
Biochemistry 35; 467-474 (1997).



From: Barbara Moffatt moffatt at sciborg.uwaterloo.ca
Jagreet:

I have had good success at this using the facilities of the Alberta
Peptide Institute. They have proprietary software that analyzes the
proteins and predicts the best peptides based on several criteria.
They also make the peptides conjugated to KLH. I have always had
success with the peptides they have designed and made. You can
contact them at Dept of Biochemistry, University of Alberta, Fax
403-492-7168. The person I spoke with was Dr. Robert Parker. His
phone number is 403-492-3907. He has email but I don't have it handy.
Likely you can get it from the U of Alberta web page.

Kind regards
Barb Moffatt


"Immink, R." R.Immink at plant.wag-ur.nl
There is a company in Belgium (Eurogentec) which provides these kind of
services. You can send your protein sequence and they will select the best
domains for raising an antibody. You can select yourself one of these
domains and let them make the antibody against it. You can have a look at
their website for more info:
http://www.eurogentec.be/

Good luck,


From: Francois Ouellet ouelletf at EM.AGR.CA

Here are a few points that you should look for. Most of the information
comes from "Current Protocols in Protein Science" and "Antibodies", two
very well known lab protocols books. The region to choose as an epitope
should be like this:
. of course, it has to be totally different from the other members of the
family
. 10 amino acids or longer; 12-15 residues long is good
. better to have more than one peptide for the protein
. better to have a couple of 12 a.a. peptides than only one of 25-30 a.a.
. highly hydrophilic
. highly mobile (containing glycine and proline residues)
. best if it is at the C-terminus
. you have to add a cysteine residue at the N-terminus if the peptide is
to be
coupled to the carrier using MBS
   * in this case, there should not be a cysteine residue within your
peptide

You can check the following web site to find programs to analyze your
protein.This is the reference site for protein work.

http://www.expasy.ch

Another possibility would be to analyze your protein using the
"peptidestructure" command on the GCG software package from the University
of
Wisconsin, if you have access to it.

I had an antibody made against an internal peptide of a family of over 20
members. I was successful in generating an antibody against a peptide of
12amino acids (13 with the cysteine), coupled to the carrier KLH using MBS
as acoupling agent. I had the antibody made at Genosys, a company in the USA.
They did everything after I sent them the sequence of the peptide: peptide
synthesis, coupling to KLH, injections in rabbits, blood collection. The
antibody was excellent for western blot analysis but showed a very low
efficiency in immunoprecipitation. You may want to use a few peptides if
you are to do IP. The mixture of peptides can be injected in the same
rabbit.
Theonly thing is that the cost of peptide synthesis is prohibitive.

I hope this will help. Cheers.

Francois
Francois Ouellet
Agriculture and Agri-Food Canada



From: Mike Mulligan rmmullig at uci.edu
We have made several anti-peptide antibodies, take a look at these papers:
rps12 Transcripts Results in the Synthesis of Polymorphic Polypeptides in
Plant
Mitochondria.  The Plant Cell 8:107-117

Williams, M.A., Tallakson, W.A. Phreaner, C.G., and R.M. Mulligan (1998)
Editing and Translation of Ribosomal Protein S13 Transcripts: Unedited
Translation Products Are Not Detectable in Maize Mitochondria.  Current
Genetics 34:221-226

Let ne know if you have other questions.

Mike



From: Jon Monroe monroejd at jmu.edu

I recently went through the same process that you are going through
and found lots of good information at
http://www.genosys.com/index2.html.  They (Sigma Genosys) were quite
helpful and I'm looking forward to getting the antibodies in a few
weeks.

Best of luck,

Jon


"Jerry D. Cohen" cohen047 at tc.umn.edu
We have done this fairly often through Research Genetics
(http://www.resgen.com/).  There are a lot of different things to
consider,but they are good at walking you through the details.  Of 
course you need
to decide if you want to pick up the whole family or design a specific
antibody to your family member, etc.  Hope this helps, Jerry

Dr. Jerry D. Cohen
Professor, Bailey Endowed Chair
Department of Horticultural Science
University of Minnesota
1970 Folwell Ave., 305 Alderman Hall



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