The Li-Cor In-Cell Western (ICW) assay, which uses infrared-labelled secondary antibodies to detect phospho-ERK, is suited for drug discovery applications where protein quantification is important in the cellular environment. This includes IC50/EC50 determination, GPCR trafficking and/or function and RNAi screens.
In addition the assay is suited for drug discovery assay development, secondary screening and lead optimisation labs. The assay enables compounds to be filtered out faster, allowing an emphasis on valid hits.
The paper, published in Analytical Biochemistry and entitled: "A 384-well cell-based phospho-ERK assay for dopamine D2 and D3 receptors," details the use of a 384-well, cell-based functional assay to quantify dopamine agonist-induced ERK phosphorylation in D2- and D3-overexpressed cell lines.
Dr. Michael Olive, Li-Cor vice president of research and development said: "Comparing the phospho-ERK ICW to other standard assays of GPCR function, neither agonist-induced calcium mobilization, as monitored by FLIPR, nor agonist-mediated inhibition of adenyl cyclase was observed yet dopamine-induced phospho-ERK was elevated five- to six-fold demonstrating the sensitivity of the phospho-ERK ICW."
The In-Cell Western assay is a faster method for analysing signal transduction pathways. The assay can quantify proteins in a 96- or 384-well microplate in less time than a gel-based Western. In-Cell Westerns use infrared dye-labelled secondary antibodies to directly label proteins in cells.
The Odyssey Infrared Imaging System, which quantifies the signals in the wells of the microtitre plate, is equipped with two infrared channels for direct fluorescence detection on membranes with sensitivity that rivals chemiluminescence. The signal-to-noise ratio produced by the near infrared dyes produces results not possible with traditional fluorescence-based imaging systems.
A spokesman for Li-Cor biosciences told DrugResearcher.com: "Although there are assays used in drug discovery to analyse signal transduction pathways, they are limited as they only provide a single data point (i.e. single protein in pathway) and they do not represent the true cellular environment."
Currently visible fluorescent detection systems on the market are limited in sensitivity due to the high amount of background created by biomolecules and plates fluorescing in the visible region.
"Infrared detection does not have these limitations as biomolecules and other media (plates, membranes) do not fluoresce in the infrared region. This results in high sensitivity with a large linear range of detection," he said