By combining strong in-house knowledge of chemistry, immunology, and signal transduction with dedicated R&D and production, Life Technologies is your first choice for cell biology research.  We offer more than 300 Novex® phosphorylation site–specific antibodies (phosphospecific antibodies) and is actively expanding this product line to enable analysis of key targets in normal and disease states, including cardiovascular disease, cancer, inflammation, neurodegenerative diseases, and diabetes. These new tools are providing critical insight into the investigation of complex signal transduction events, including unraveling the intricacies of drug intervention in disease.

  • Confidence—extensive validation ensures highly specific antibodies
  • Performance—robust antibodies validated for multiple applications
  • Selection—large menu of unique specificities

View all phosphospecific antibodies

What are phosphospecific antibodies?


Phosphospecific antibodies are affinity-purified rabbit polyclonal or monoclonal antibodies that are monospecific for a target protein that is phosphorylated on a specific tyrosine, threonine, or serine residue (Figure 1).

Figure 1—Detection of protein phosphorylation.
Detection of dual phospho-ERK1/2 in insulin-treated 3T3L1 adipocytes. J Biol Chem (1998) 273:31408. Courtesy of Dr. Jacqueline Stephens, Louisiana State University.

Why use Novex® phosphospecific antibodies?

Our phosphospecific antibodies:

  • Allow the status of (an) individual phosphorylation site(s) to be easily determined, which can provide an indication of the active state of a protein (Figure 1)
  • Can be used in a wide variety of immunodetection applications includine immunohistochemistry, quantitative ELISA and flow cytometry (Figure 2)
  • Allow the subcellular localization of the phosphorylated protein to be studied by immunostaining

IHC analysis of ERK1/2

Figure 2A. 
Immunohistochemical analysis of ERK1/2 phosphorylation in mouse heart sections from normal (inset) or cardiomyopathic animals using ERK1/2 [pTpY185/187] phosphospecific antibody.

ERK1/2 in Jurkat cells

Figure 2B.  Quantitative ELISA analysis of phosphorylated ERK1/2 in Jurkat cells using  ERK1/2 [pTpY185/187] phospho- specific antibody.

Figure 2C. Flow cytometric analysis of ERK1/2 phosphorylation in PMA-treated Jurkat cells using ERK1/2 [pTpY185/187] phosphospecific antibody. Generated with cell extracts kindly provided by Dr. Michael  Schaller, University of North Carolina.

How are our 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 (Figure 3)

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

 Phosphospecific Purification Process
  Figure 3—Purification process. Lane 1: generic anti-pY; lane 2: anti–Ron [pYpY1330/1337] after positive [pYpY1330/1337] peptide purification; lane 3: anti–Ron [pYpY1330/1337] after positive [pYpY1330/1337] peptide and negative generic pY peptide purification. The data demonstrate the effectiveness of combining negative and positive affinity purification.

How is antibody specificity demonstrated?

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

  • Western blot analysis in multiple cell lines
  • Peptide/phosphopeptide competition experiments (Figure 4)
  • Analysis of site-directed mutants (Ser/Thr→Ala or Tyr→Phe) (Figure 5)

FAK specificity

Figure 4. Specificity.
FAK [pY397] phosphospecific antibody analysis of CEF cells illustrate that only the phosphopeptide corresponding to FAK [pY397] blocks the western blot signal. Generated with cell extracts kindly provided by Dr. Michael Schaller, University of North Carolina.

FAK phosphospecific

Figure 5A. 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.

Signal to Noise

Figure 5B. 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 about the QC of our 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, including both in-house and beta-site testing
  • Demonstration of lot-to-lot consistency
  • Combination of 2 or more pre-qualified individual lots to create each batch of antibody, thereby minimizing batch-to-batch variability

The Novex® development approach greatly increases the chances of generating antibodies with:

  • High-(mono) specificity for the target of interest
  • Excellent lot-to-lot consistency
  • High-affinity recognition
  • High-avidity reactivity
  • Superior signal-to-noise ratios
  • Ability to perform in a variety of immunodetection applications 

Novex® Antibodies versus the competition

PC12 cells

Novex®’ stringent quality control specifications and proprietary manufacturing methods result in antibodies that possess superior sensitivity relative to competitors. The high-avidity reactivity of our antibodies often allows detection of endogenous protein levels in cell extracts (Figure 6).

Figure 6. Novex® phosphospecific antibodies. Untreated PC12 cells were compared to cells treated with sorbitol, using antibodies against p38. Western blotting was performed using manufacturer protocols supplied with each product.