Generating polyclonal antibodies from peptides for ChIP-Seq

sghussey · 02-09-2009, 10:23 AM

My question pertains to the generation of polyclonal antibodies from synthetic peptides, such as those provided by GenScript, for use in ChIP-Seq. I recognize a potential problem in submitting a single peptide for antibody generation: that the specific protein epitope recognized by the antibody and represented by the synthetic peptide will not be exposed after formaldehyde cross-linking of the protein-DNA complex. Would it therefore be possible to obtain a polyclonal antibody by inoculating rabbits with two synthetic peptides from the same protein, such that more than one epitope may be recognized by the antibody?

EPICENTRE · 03-04-2009, 08:17 AM

I don't think the 2-peptide strategy will work because each of the peptide will be independently displayed by the immune system. I know of a few companies that are trying this approach (with a single peptide) and are having marginal success.

If you are married to using a peptide antigen, you may want to try raising a monoclonal so you can screen for the desired properties (recognizing DNA-bound protein). Here though, the major challenge is having target to use in your screening.

Another thought would be to try a genetic immunization approach (like SDI). I've never tried them, but it looks look on paper...

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Ion Sphere ID summary, polyclonal %	The_Dark_Base	Ion Torrent	3	12-23-2011 01:08 PM
ChIP-Seq: ChIP-Array: combinatory analysis of ChIP-seq/chip and microarray gene expre	Newsbot!	Literature Watch	0	05-19-2011 03:50 AM
ChIP-Seq: ChIP-chip versus ChIP-seq: Lessons for experimental design and data analysi	Newsbot!	Literature Watch	0	03-02-2011 03:50 AM
Non-specific binding of control antibodies for ChIP-seq	jdub	Epigenetics	1	12-01-2010 09:20 PM
ChIP-Seq: ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip dat	Newsbot!	Literature Watch	0	05-13-2010 03:00 AM

02-09-2009, 10:23 AM	#1
sghussey Junior Member Location: Pretoria, South Africa Join Date: Feb 2009 Posts: 2	Generating polyclonal antibodies from peptides for ChIP-Seq My question pertains to the generation of polyclonal antibodies from synthetic peptides, such as those provided by GenScript, for use in ChIP-Seq. I recognize a potential problem in submitting a single peptide for antibody generation: that the specific protein epitope recognized by the antibody and represented by the synthetic peptide will not be exposed after formaldehyde cross-linking of the protein-DNA complex. Would it therefore be possible to obtain a polyclonal antibody by inoculating rabbits with two synthetic peptides from the same protein, such that more than one epitope may be recognized by the antibody?

03-04-2009, 08:17 AM	#2
EPICENTRE Registered Vendor Location: Madison Join Date: Mar 2009 Posts: 5	I don't think the 2-peptide strategy will work because each of the peptide will be independently displayed by the immune system. I know of a few companies that are trying this approach (with a single peptide) and are having marginal success. If you are married to using a peptide antigen, you may want to try raising a monoclonal so you can screen for the desired properties (recognizing DNA-bound protein). Here though, the major challenge is having target to use in your screening. Another thought would be to try a genetic immunization approach (like SDI). I've never tried them, but it looks look on paper...