Phosphorylation is the addition of phosphate groups to proteins. Phosphate groups attach to a serine, threonine, or tyrosine residue via a kinase mediated reaction. Phosphorylations are important for activation of various pathways. Phosphospecific antibodies are affinity-purified rabbit polyclonal or monoclonal antibodies that are monospecific for a target protein that is phosphorylated. These antibodies provide critical insight into the investigation of complex signal transductions events.

Why use phosphospecific antibodies?

Phosphospecific antibodies allow the status of individual phosphorylation site(s) to be easily determined, which can provide an indication of the active state of a protein.They are widely used in a variety of immunodetection applications including western blot (figure 1), immunohistochemistry (figure 2), and flow cytometry (figure 3).

Figure 1. Altered expression of dual phosphorylation of ERK1/2 upon different treatments in various cell lines. Phospho-ERK1/2 (Thr202, Tyr204) Monoclonal Antibody was used (Product # 14-9109-82).
Immunohistochemical analysis of ERK1/2
Figure 2. Immunohistochemical analysis of formalin-fixed paraffin embedded human breast cancer tissue using Phospho-ERK1/2 (Thr202, Tyr204) Monoclonal Antibody (Product # 14-9109-82).
Flow cytometry analysis of ERK1/2
Figure 3. Flow cytometric analysis of Phospho-ERK1/ERK2 (Tyr202/Tyr204) in U0126-inhibited (blue) or PMA-stimulated (green) Jurkat cells using a Phospho--ERK1/ERK2 (Tyr202/Tyr204) monoclonal antibody (Product # MA5-15174) compared to a nonspecific negative control antibody (red).

How are Invitrogen phosphospecific antibodies made?

We use a series of proprietary manufacturing methods including:

  • Antigen design and presentation
  • Multiple immunization strategies
  • Extensive affinity purification using both positive and negative adsorption methodologies to optimize the desired reactivity 

The result is the generation of phosphospecific antibodies that produce a robust signal and are highly specific to the target protein.

How are Invitrogen phosphospecific antibodies made?

Every effort is made to ensure that the polyclonal antisera are monospecific for the targeted phosphorylated sequence. QC data are included in every antibody data sheet. Our specificity testing includes:

  • Western blot analysis in multiple cell lines
  • Non-phosphopeptide/phosphopeptide competition experiments (figure 4)
  • Analysis of site-directed mutants (Ser/Thr→Ala or Tyr→Phe) (figure 5)
Western blot analysis of ERK1/2
Figure 4. Phospho-ERK1/ERK2 (Thr185, Tyr187) Antibody (Product # 44-680G) analysis of sorbitol-stimulated PC12 cells illustrate that only the phosphopeptide corresponding to ERK1/2 (pT185/pY187) blocks the western blot signal. The signal cannot be blocked by generic pT or pY peptide nor the non-phosphorylated ERK1/2 (T185/Y187) peptide.
FAK [pY397] phosphospecific antibody analysis illustrating the absence of a western blot signal with the FAK (Tyr→Phe F397) mutant
Figure 5. Superior signal-to-noise ratios. FAK [pY397] phosphospecific antibody analysis illustrating the absence of a western blot signal with the FAK (Tyr→Phe F397) mutant.

What quality control testing is done on Invitrogen phosphospecific antibodies?

We have a rigorous and routine manufacturing process including:

  • Utilization of standardized procedures for phosphospecific antibody manufacturing along with appropriate controls in all testing as an integral part of our quality assurance program
  • Validation in multiple cell lines and species
  • Demonstration of lot-to-lot consistency

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