Generation, purification, and identification of phosphospecific rabbit anti human CXCR4 polyclonal antibody

Phosphorylation on the Serine, Threonine and/or Tyrosine residues is an important even of post-translational modification of proteins essential to correct functioning within cells. In this study, a simple method was developed to produce and to affinity purify the specific rabbit anti-phosphopeptides polyclonal antibody. Phosphopeptides crosslinked with hemocyanin of keyhole limpets (KLH) and bovine serum albumin (BSA) were used as immunogens to raise the antiserum. Phosphospecific polyconal IgGs were obtained by “two-step” peptide-affinity column purification. The specificity of isolated rabbit IgGs was tested by indirect ELISA and Western blot, respectively. This immune method also can be used to produce anti-methylspecific and anti-acetylspecific protein polyclonal or monoclonal antibody.