A rabbit phospho-specific polyclonal antibody was generated by Abmart using the phosphopeptide C-QFDR(pS)FILAS encompassing the S269 site in SYP-4. Two peptides were synthesized: An antigen peptide with phosphoserine C-QFDR(pS)FILAS and a control peptide without phosphorylation (C-QFDRSFILAS). Serum harvested from the rabbits immunized with the antigen peptides went through two rounds of affinity purification. First, serum was passed through a column to which the antigenic peptide was coupled in order to isolate phospho-specific SYP-4 antibodies. The eluate from the first column was then passed through a second column to which the control peptide was coupled and flow through was collected. This second step was done to remove any non-phosphorylated SYP-4 antibodies. An ELISA titer of ≥ 1:50,000 against the modified peptide and a modified/unmodified titer ratio of ≥ 8 were used as validation criteria.
syp-4