Technical Notes: phospho-specific ELISAs

Intracellular signal transduction factors, such as the MAP Kinases, have become important drug targets and experimental biomarkers. Because the activity of these factors is regulated by phosphorylation events, accurate detection of the level of phosphorylated target proteins is paramount to assessing the effects of experimental treatments. R&D Systems’ phospho-specific DuoSet® IC (Intracellular) ELISA Development Systems offer a rapid, specific, sensitive, and economical way to measure phosphorylated proteins.

To test for phospho-specificity, lysates from untreated cells and cells treated with cytokines known to cause target protein phosphorylation are examined by ELISA and Western blot using phospho-specific antibodies. For example, Figure 1 depicts p38 alpha phosphorylation as measured by R&D Systems’ phospho-p38 alpha DuoSet IC ELISA and by Western blot. The data show a clear rise and fall in phosphorylated p38 alpha with no change in the pool of total p38 alpha, confirming the phospho-specificity of the ELISA and Western antibodies. When run side-by-side with a comparable competitor’s ELISA kit (Figure 2), R&D Systems’ phospho-p38 alpha DuoSet IC ELISA kit performed with much greater phospho-specificity.

Figure 1. DuoSet IC vs. Western blot
HepG2 cells were cultured with IL-1 beta (10 ng/mL) to induce phosphorylation of p38 alpha. Lysates from successive time points were analyzed by R&D Systems’ phospho-p38 alpha rises and then falls, while the level of total p38 remains constant on Western blot (inset).
Figure 2. Phospho-specificity: DuoSet IC vs. Competitor
HepG2 cells were cultured with or without IL-1 beta (10 ng/mL) to induce phosphorylation of p38 alpha. Lysates were analyzed using R&D Systems’ phospho-p38 alpha observed with R&D Systems’ DuoSet IC ELISA and Western blot (inset) is consistent with high phospho-specificity while the competitor’s ELISA appears to measure both phosphorylated and unphosphorylated p38.


Phospho-specific DuoSet IC ELISA Development Kits
phospho-ERK2 DYC1483
phospho-p38 alpha DYC869
phospho-p70 S6 Kinase DYC896
phospho-RSK1 DYC892B
Phospho-specific antibodies are also available for Akt, ERK, JNK, MSK, p38 alpha, p70 S6 Kinase, and RSK.