New Western Blot for Quantifying Proteins in a Cell

A new assay for analyzing signal transduction pathways significantly increases throughput, compared to traditional Western blots, and is designed to facilitate screening of drug candidates for effects on phosphorylation.

Called In-Cell Western, the assay is performed in fixed cultured cells in 96- and 384-well microplates, bypassing the gels and cell lysates usually needed for Western blotting. The assay offers a 30-fold increase in throughput over traditional Western blots, says Li-Cor Biosciences (Lincoln, NE, USA; www.licor.com), which developed the assay. In-Cell Western assays were developed exclusively for Li-Cor's Odyssey infrared imaging system and are used to quantify proteins directly in the cell. Odyssey uses two-color detection to simultaneously quantify two targets, which increases accuracy by providing easy data normalization.

The assay is particularly powerful for quantitative assessment of phosphorylation with phospho-antibodies, and was designed to aid the screening of drug candidates for phosphorylation effects. The Odyssey system images infrared dye-labeled secondary antibodies to detect proteins in cells, and fluorescence from each well in the microplate is quantified.

"Quantification accuracy is enhanced and data are more meaningful since proteins are detected in their cellular context,” said Dr. Amy Geschwender, Li-Cor's manager of molecular biology.




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