Firefly 3000: a Western blot and more — in a capillary

The Firefly^™ 3000 protein analysis system enables quick and quantitative assays of low-abundance proteins. Now you can plan and execute experiments with extremely small samples (as few as 25 cells), and get detailed information on the phosphorylation status of critical signaling proteins. The system's automation speeds samples from a standard 96-well microplate to results, ensuring exceptional sample-to-sample consistency as well as convenience.

Firefly assays provide information similar to a Western blot, but are performed in an automated, capillary-based instrument. As in Western blot analysis, proteins from complex biological samples are separated, immobilized, and probed with antibodies. However, the Firefly system uses an IEF separation to resolve the various phosphorylation states of signaling proteins. The separated proteins then are immobilized to the capillary wall and probed with primary and secondary antibodies. The secondary antibody is HRP-labeled, which enables ultrasensitive chemiluminescence detection. Even low-abundance proteins can be rapidly measured in tiny samples.

The figure below demonstrates how assays are developed for the Firefly 3000. In this example, the targets are ERK1 and ERK2. First, we choose a relevant biological model system in which the target proteins can be induced. We then run the non-induced and induced cell lysates on the Firefly 3000 and probe different capillaries with antibodies to the various phosphorylation states of the target protein. Peaks are identified by correlating the results from multiple antibodies. Once the peaks have been identified, the routine assay can be run using only a single pan-specific antibody.

Western blot and Firefly analyses of ERK1 and ERK2 isoforms in cell lysates from HT-29 cells treated with insulin and TNF-alpha. Conventional SDS/PAGE Western blots were probed with antibodies specific for pan-ERK (ERK1/2), ERK1, ERK2 and phosphorylated ERK1 and 2 (pERK1/2). Samples shown are from insulin and TNF-alpha treatment for 0 min (black box) and 30 min (red box). Firefly analyses of cell lysates were probed with the same antibodies (listed on the traces): untreated 0 min (top trace), and 30 min insulin and TNF-alpha treatment (lower four traces). Probing with ERK antibodies with different specificities allowed the peak identifications, as noted.