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peptide design for immunization


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#1 andrcc

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Posted 13 May 2011 - 07:27 AM

hi, im new to this...

so if im going to design peptides for raising antibodies. what are the standard procedures? are there any free softwares (i do found paid version by google, but i dunt think my boss is going to pay for it.)

many thanks in advance

#2 BioMiha

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Posted 13 May 2011 - 10:27 AM

Depends on what type of Abs do you want. However in my experience if you want to get Abs that crossreact with a native protein, there is a very high chance the peptide immunization approach won't work. If you provide some more info, you might get a bit more feedback. Otherwise there is not much in the way of software, so the payable services are really not worth the buck.

#3 andrcc

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Posted 13 May 2011 - 11:14 PM

Depends on what type of Abs do you want. However in my experience if you want to get Abs that crossreact with a native protein, there is a very high chance the peptide immunization approach won't work. If you provide some more info, you might get a bit more feedback. Otherwise there is not much in the way of software, so the payable services are really not worth the buck.


thanks biomiha,

what we need is ab for western/ ICC. but we dunt have the recombinant proteins yet. (and there are quite a number of genes to clone)
so the plan is to raise ab by synthetic peptide, along with the cloning process.
any comment/ recommendation would be much appreciated!

#4 BioMiha

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Posted 14 May 2011 - 12:27 AM

We've done a lot of peptide immunizations over the years and obtaining anti-peptide Abs is not a problem, but obtaining Abs that crossreact with the native protein is nearly mission impossible. If you check with commercial services that can provide you with for example rabbit sera after peptide immunization, they will always state that they don't guarantee protein crossreactivity. If you want to read the theory behind this I recommend one of Marc Van Regenmortel's review papers.
So, that being said, I would definitely try to express (or buy if you can) the recombinant protein and go from there. If as you said you can't do that, I can give a few hints about how to choose the peptides for immunization.
1. You want the peptide to be a part of a surface exposed region of your protein. There is no use immunizing with a peptide that will be buried in the protein, because the Abs won't be able to bind.
2. A good idea is to search for loops, because you have a higher chance of getting crossreactivity.
3. Do a BLAST run with the peptide you designed so you get an idea how many and what kind of proteins the Abs could bind to. If there is considerable crossreactivity in the system you will use, you will have to redesign the peptide.
4. Be sure to block the free ends of the peptide so they don't carry a charge - the charged ends are highly immunogenic and you will get Abs to these instead of your protein.
5. If your peptide is shorter than say 20 a.a. (and it should be because longer peptides may form a stable secondary structure) you need to couple it to a carrier protein e.g. KLH, which is a science all on it's own. The best thing to do is to add an N-terminal cysteine to the peptide so you can couple via the SH-group. Other coupling/conjugation options are much worse.
6. Pray to the gods of peptide immunization that you will get Abs that bind your native protein. Some people say that offering them high value possessions makes them happy and perhaps they will be merciful.

Edited by BioMiha, 14 May 2011 - 12:29 AM.


#5 andrcc

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Posted 15 May 2011 - 08:03 AM

We've done a lot of peptide immunizations over the years and obtaining anti-peptide Abs is not a problem, but obtaining Abs that crossreact with the native protein is nearly mission impossible. If you check with commercial services that can provide you with for example rabbit sera after peptide immunization, they will always state that they don't guarantee protein crossreactivity. If you want to read the theory behind this I recommend one of Marc Van Regenmortel's review papers.
So, that being said, I would definitely try to express (or buy if you can) the recombinant protein and go from there. If as you said you can't do that, I can give a few hints about how to choose the peptides for immunization.
1. You want the peptide to be a part of a surface exposed region of your protein. There is no use immunizing with a peptide that will be buried in the protein, because the Abs won't be able to bind.
2. A good idea is to search for loops, because you have a higher chance of getting crossreactivity.
3. Do a BLAST run with the peptide you designed so you get an idea how many and what kind of proteins the Abs could bind to. If there is considerable crossreactivity in the system you will use, you will have to redesign the peptide.
4. Be sure to block the free ends of the peptide so they don't carry a charge - the charged ends are highly immunogenic and you will get Abs to these instead of your protein.
5. If your peptide is shorter than say 20 a.a. (and it should be because longer peptides may form a stable secondary structure) you need to couple it to a carrier protein e.g. KLH, which is a science all on it's own. The best thing to do is to add an N-terminal cysteine to the peptide so you can couple via the SH-group. Other coupling/conjugation options are much worse.
6. Pray to the gods of peptide immunization that you will get Abs that bind your native protein. Some people say that offering them high value possessions makes them happy and perhaps they will be merciful.


thats a lot of information. thanks a lot biomiha :D
and then i'll strat to pray day and night

#6 bob1

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Posted 15 May 2011 - 04:25 PM

check out the following URLs for linear B-cell peptide prediction:

http://www.imtech.re...xpred/link.html

http://ailab.cs.iast...ds/predict.html

http://www.immuneepitope.org/links.php

http://immunopred.or...inks/index.html




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