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.
What You Need To Know About 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.
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
Figure 2B. Quantitative ELISA analysis of phosphorylated ERK1/2 in Jurkat cells using ERK1/2 [pTpY185/187] phospho- specific antibody.
We use a series of proprietary manufacturing methods including:
|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.|
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)
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®’ 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.