Production of motif-specific and context-independent antibodies using peptide libraries as antigens
First Claim
1. A method for producing a motif-specific, context-independent antibody that specifically binds a recurring, modified motif in a plurality of peptides or proteins within an organism in which it recurs, said method comprising the steps of:
- (a) constructing a degenerate peptide library comprising (i) a fixed target motif comprising two to six invariant amino acids including at least one modified amino acid, and, optionally, one or more degenerate amino acid position(s), and (ii) a plurality of degenerate amino acids flanking said motif;
(b) immunizing a host with said peptide library to raise a context-independent antibody specific for all or part of said target motif; and
(c) isolating antisera from said host, and purifying the motif-specific, context-independent antibody of step (b) from said antisera, said antibody specifically binding said motif in a plurality of peptides or proteins within an organism in which it recurs.
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Abstract
A method is provided for producing motif-specific, context-independent antibodies which recognize a plurality of peptides or proteins within a genome that contain the same motif. The method includes the step of immunizing a host with a degenerate peptide library antigen featuring (i) a fixed target motif containing one or more invariant amino acids including at least one modified amino acid, and (ii) a plurality of degenerate amino adds flanking the motif. Motif-specific, context-independent antibodies produced by the disclosed method are also provided. The method encompasses motifs consisting of a single modified amino acid, as well as short motifs comprising multiple invariant amino acids including one or more modified amino acids, such as all or part of kinase consensus substrate motifs, protein-protein binding motifs, or other cell signaling motifs. Methods of using the antibodies, e.g. for genome-wide profiling, are also provided.
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Citations
14 Claims
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1. A method for producing a motif-specific, context-independent antibody that specifically binds a recurring, modified motif in a plurality of peptides or proteins within an organism in which it recurs, said method comprising the steps of:
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(a) constructing a degenerate peptide library comprising (i) a fixed target motif comprising two to six invariant amino acids including at least one modified amino acid, and, optionally, one or more degenerate amino acid position(s), and (ii) a plurality of degenerate amino acids flanking said motif; (b) immunizing a host with said peptide library to raise a context-independent antibody specific for all or part of said target motif; and (c) isolating antisera from said host, and purifying the motif-specific, context-independent antibody of step (b) from said antisera, said antibody specifically binding said motif in a plurality of peptides or proteins within an organism in which it recurs. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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- 10. A motif-specific, context-independent antibody that specifically binds a recurring, phosphorylated motif consisting of (i) two to six invariant amino acids including at least one phosphorylated amino acid, and, optionally, (ii) one or more degenerate amino acid position(s), said antibody specifically binding said motif in a plurality of non-homologous peptides or proteins within an organism in which it recurs.
Specification