A phospho-specific antibody makes a demanding claim: that it recognizes a sequence only when one particular residue carries a phosphate group. Demonstrating that claim is largely a matter of having the right peptides, and the reagents needed are simple enough that there is little excuse for omitting them. This page describes the peptide set and the experiments that use it.
Everything rests on two peptides that are identical except for the phosphate group.
Synthesize both in the same batch, to the same purity, with the same terminal chemistry and the same tags. A pair made months apart by different routes is a weaker control, because any difference in behaviour has an alternative explanation. If the antibody will be used against a site that sits near other phosphorylatable residues, consider adding peptides phosphorylated at those neighbouring positions as well; this is how cross-reactivity within a cluster is detected.
Coat or spot a dilution series of each peptide and probe with the antibody. A specific antibody gives a strong signal on the phospho peptide and little or none on the non-phospho peptide across the range. Reporting the ratio at matched peptide amounts is more informative than a single concentration, because weak cross-reactivity only becomes visible at higher loading.
This is the experiment that carries the most weight, because it tests the antibody in the context where you actually use it. Pre-incubate the antibody with excess free phospho peptide and run the Western blot, immunofluorescence or immunohistochemistry as usual; the specific signal should disappear. Pre-incubate a parallel aliquot with the same excess of non-phospho peptide; the signal should remain.
Both halves matter. A signal blocked by the phospho peptide alone shows that it depends on that sequence. A signal that survives the non-phospho peptide shows that the dependence is on the phosphate, not merely on the surrounding residues.
Treat one half of a lysate or a fixed sample with a phosphatase and leave the other half untreated under the same conditions. A genuine phospho-specific signal is lost in the treated sample. This control is valuable because it works on the biological material rather than on peptides, and it catches antibodies that are specific against synthetic peptides yet promiscuous against a complex sample.
Stimulate or inhibit the pathway that phosphorylates the site and show that the signal moves in the expected direction. Where a phospho-null mutant such as a serine-to-alanine substitution can be expressed, loss of signal in that background is strong evidence. Genetic controls of this kind are increasingly expected for antibodies used in a central claim.
Many kinase substrate motifs recur across the proteome, so an antibody raised against one phosphosite may recognize others. Screening against a panel of related phospho peptides, including the neighbouring sites in the same protein, puts a boundary on the claim. A result stating which related sequences were tested and not recognized is far more useful to a reader than an unqualified assertion of specificity.
For a polyclonal antiserum raised against a phospho peptide, purification largely determines how specific the final reagent is. The usual approach is two steps: positive selection on a column carrying the phospho peptide, then depletion on a column carrying the non-phospho peptide to remove antibodies that bind the sequence regardless of modification. Antibodies that survive both steps are the ones worth validating.
This is why the peptide order for a phospho antibody project is larger than for a standard one. Besides the immunogen, you need enough of both peptides to prepare two affinity columns and to run the validation. Planning that at the start avoids a pause between immunization and usable reagent.
Phospho and sulfo are nearly the same mass
Phosphorylation adds 79.97 Da and sulfation 79.96 Da, a difference of about 0.01 Da. On a low-resolution mass spectrometer the two are indistinguishable. If you work with tyrosine modifications and the analysis matters, use a high-resolution instrument and say which one was used. See our modification mass table.
Paste a peptide or protein sequence to review length, charge, hydrophobicity and other properties before you order.
Research use only. LifeTein peptides and proteins are supplied for laboratory research use. They are not for human or veterinary use, and not for diagnostic or therapeutic use. See our research use policy.
We can supply matched phospho and non-phospho peptide pairs, carrier conjugates and affinity columns as one package, and run the antibody project alongside them. Email sales@lifetein.com or use our antibody service quotation form.