Protein or peptide for Ab production?
cfritze at ix.netcom.com
Tue Nov 24 11:45:19 EST 1998
I'll make a short contribution. Others will surely weigh in on the points I
An antibody raised against the full-length protein or sub-domains will be
more generally useful. Your ability to predict which peptides will be (1)
antigenic, and (2) in functionally significant regions of the protein are
limited at this point. Also, polyclonal sera raised against a larger portion
of the protein are more likely to function in immunohistochemistry than an
anti-peptide antibody since the polyclonal sera will be active against a
range of epitopes, at least some of which will be accessible for staining.
And finally, once you learn more about the protein and desire
region-specific antibodies you can affinity purify this serum against
further sub-regions of the protein you immunized with.
Anti-peptide antibodies have the advantage of being cost/labor saving to
generate (no making expression vector, fooling with expression conditions,
purification, etc.) Furthermore, if a run through GenBank indicates that
much of this protein is highly similar to other proteins in your system,
judicious selection of a peptide sequence can give you a more specific
antibody than you would get by using a full-length immunogen. And finally,
there are times when it comes in handy to know which epitope is being
recognized by your antibody without having to do additional experiments.
Christian Fritze, Ph.D.
Director of Technology Transfer
Covance Research Products,
formerly Berkeley Antibody Company (BAbCO), same quality antibodies and
christian at babco.com
(510) 412-8930, -8940 fax
"Erkki Kuusisto" <ekuusist at JALUS.UKU.FI> wrote in message
2A55DAF3337 at jalus.uku.fi...
>What is the method of choice to prepare an antigen for antibody
>production, starting from cDNA sequence? I have the cDNA of a protein
>for which I would like to make Westerns and, possibly,
>immunohistochemistry. A commercial Ab is not yet available. The
>protein is from rat, 55 kD, likely soluble and cytoplasmic, and
>contains several putative phosphorylation sites.
>Is it best to express in bacteria the protein with a peptide tag
>sequence, and affinity purify the tagged protein? If so, which
>purification tag would be most practical?
>Or, for best results, am I better off using a synthetic peptide as
>the antigen instead of the complete protein?
>Any suggestions would be helpful. Thank you in advance.
>University of Kuopio
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