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Do all cell-based kinase assays perform similarly? A side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen Surefire

Charrier-Savournin F, Vallaghé J, Costy N, Douzon S, Chouvet C, Boisseau C, Degorce F, Trinquet E

2013

Cisbio Bioassays, Codolet, France

SLAS 2013, Orlando, Florida, USA

Learn how HTRF performed for phospho-STAT3, -p38 MAPK, -IKKb, -mTOR, -CREB, -MEK1.

Functional responses induced by pharmacological compounds can be assessed through the quantification of specific biomarkers. Among them, protein phosphorylation is a ubiquitous readout reporting for various biological responses such as mitogenstimulated proliferative effect and inflammatory response upon cytokine exposure, as well as metabolic response following hormonal treatment. HTRF cellular kinase assays are intended to measure the phosphorylated status of a protein in a cellular context. The detection is achieved using a pair of antibodies labeled with HTRF probes: a first antibody raised against the phosphorylation residue of interest, and the second raised against the total protein. If the protein of interest becomes phosphorylated, a time-resolved FRET process will occur between the 2 labeled antibodies (scheme 1: assay principle & protocol). In this study, the performances of different HTRF cellular kinase assays were benchmarked against different technologies commonly used in the kinase research field, such as Western Blot, Elisa and Surefire. This poster provides comparative data on the following assays: p-IKKb, p-Stat3, p-p38 MAPK, p-mTor and p-Creb, with a particular emphasis on p-IKKb.

Cellular phospho-/total protein assays, Kinases & cellular phospho-/total protein assays





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