Antibodies that recognize intact proteins can be produced through the use of synthetic peptides based on short stretches of the protein sequence, without first having to isolate the protein. The steps to produce an effective antibody include: (1) designing the peptide sequence based on the sequence of the protein; (2) synthesizing the peptide; (3) preparing the immunogen either by coupling the synthetic peptide to a carrier protein or through the use of a multiple antigenic peptide (MAP); (4) immunizing the host animal; (5) assaying antibody titer in the host animal's serum; and (6) obtaining the antiserum and/or isolating the antibody. This unit covers steps 1 and 3. Once the coupling procedure has been performed, it is possible to determine the approximate degree of coupling by amino acid analysis. Also presented are methods for assaying free sulfhydryl content and for reducing disulfide bonds in synthetic peptides.
|Pages (from-to)||Unit 9.2|
|Journal||Current protocols in immunology / edited by John E. Coligan ... [et al.]|
|State||Published - Aug 2003|