polyclonal antibodies: Help!!
sferguso at kimbark.uchicago.edu
Mon Sep 25 07:43:21 EST 1995
In article <445aml$dds at galactica.galactica.it>,
Giorgio Spagnol <spagnol at galactica.it> wrote:
>Dear fellow researchers,
>I need to raise antibodies to a type 1 membrane glycoprotein of 100 kD.
>Since I want these Ab's to act against the extracytoplasmatic peptidic
>moyety, and I do not have sufficient quantity of purified antigen, I
>tought about using a sinthetic peptide as an immunogen.
>Do you have any idea how to choose it, given that the whole sequence of
>the protein is known?
>Thanks in Advance.
I know there are general rules for choosing them, but I don't remember
what they are. you might do best to run a medline search or consult
a manual on generating monoclonal antibodies. There are also other
tricks you could use to enhance the response, including conjugation
of the peptides to a larger carrier protein. The reason for this is
that T cells recognize small peptides. If you use only your peptide
for immunization, you'll be stimulated a very limited pool of
T cells which in turn, results in limited B cell activation (remember
that T cells are required for most B cell responses.)
By conjugating your peptide to a larger protein, you'll be evoking
a larger repertoire of T cell help, which in turn results in more
B cells being activated by your peptide (the T cell need not
recognize the same region of a moleculte as the B cell does).
Also, if you plan on using a peptide, don't expect to get a very
polyclonal response. The shorter your protein, the more limited the
range of specific B cells (as a general rule).
If I were you, I'd either subclone a fragment into an expression
vector to increase your protein production or use PCR to generate
that a fragment that contains the domain you want your antibodies
to be specific for. Purifying the protein and then taking a big
loss in chopping it up and purifying a fragment isn't my idea of
a good time :) Better to let the E coli do the work by making
gobs of pre-chopped up proteins for me.
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