Table 3.

Lessons learned and successful strategies for selection of PSSA antibodies. This table provides strategies to other researchers interested in developing phosphorylation state- and sequence specific antibodies.

Guidelines for the selection of phospho site specific antibodies from naïve antibody libraries
Do not use phosphate buffered saline
Use a combination of phage and yeast display
Biotinylate the target peptide and select in the presence of non-biotinylated competitor peptides (either other phosphorylated sites or non-phosphorylated peptides)
Elute with non-biotinylated target peptide
Use of excess bio-peptide to coat streptavidin magnetic beads reduces streptavidin binders
Three rounds of phage selection was ideal. Two rounds gave lower affinity antibodies. 4 or 5 rounds of selection resulted in overabundance of streptavidin binders.
Test antibody specificity for target peptide with competition assays with non-biotinylated competitors (competition assay). In this assay it is important to pre-mix the bio-peptide with the 10X excess nonbio-peptide prior to antibody addition (e.g. before adding yeast displaying scFv).
Test selected antibodies against real substrates as early as possible to avoid the selection of peptide-specific antibodies
Selection against one peptide does not guarantee non-reactivity against a different related peptide
Presence of protease and phosphatase inhibitors are crucial during cell lysate preparation
Lowering/absence of recognition with phosphatase treated samples is an excellent test for antibody specificity.