Mobility Shift Assays

Because of their superior data quality and target flexibility, mobility shift assays are becoming more and more used in the pharmaceutical business when used in conjunction with kinase screening assays. Creative Biolabs provides mobility shift assays to help with target selection, drug prioritization, and possible toxicity implications by enabling researchers to comprehend the general selectivity tendencies of lead compounds across the kinome.

Mobility Shift Assays in Creative Biolabs

The phosphorylated peptide substrate has a higher negative charge than the unphosphorylated form, which is exploited by the mobility shift experiment. As such, the mobility of a mixture of these peptides varies when subjected to electrophoresis. This method involves setting up tagged substrates in microplates made of polypropylene. Small quantities of each test are "sipped" up into the microfluidic chip while the kinase reaction is being conducted in the microplate. Due to variations in their velocities, the reaction's constituent parts split into substrate and product as it passes through these channels. These variations in mobility are connected to each component's charge/mass ratio. A fluorescence peak is recorded for each component as it approaches the chip's detection window.

Fig.1. Mobility shift assays.¹

Application of Mobility Shift Assays in drug discovery

Mobility shift assays are primarily used in off-chip assays for initial (hit identification) or secondary (IC₅₀ value determination) screening in the drug discovery process. Due to the technology's flexibility, assay design can be completed quickly because off-chip assays can be quickly sampled to assess enzymatic linearity. The substrate/co-factor K_mapp value may also be determined easily, and for ATP-dependent kinases, the method is not impacted by interference at high ATP levels, unlike previous methods. The mobility shift test enables the separation of product and substrate molecules according to variations in mass and/or charge. As a result, a large range of possible targets, such as protein kinases, lipid kinases, protein phosphatases, proteases, and histone deacetylases, are suitable for screening using immobility shift assays.

Fig.2. Kinase assay development.¹

Advantages of Mobility Shift Assays

When using mobility shift assays instead of alternative fluorescent approaches, there is a clear and significant advantage: the readout of the desired activity, such as (de)phosphorylation, (de)acetylation, and proteolysis, is a direct product observation. Because neither a secondary reagent nor a secondary enzyme is needed for detection, there is less chance of interfering with the tests.

Mobility shift assays are mostly used in the field of drug discovery for kinase research because of their acceptable throughput, ratio-based direct product readout, and non-radioactive nature. In this field, mobility shift assays have the potential to yield fast enough and precise results to serve as a first screening tool. Please contact us for more information about our mobility shift assays for kinase drug discovery.

Reference

1. Perrin, Dominique, Christèle Frémaux, and Adam Shutes. "Capillary microfluidic electrophoretic mobility shift assays: application to enzymatic assays in drug discovery." Expert Opinion on Drug Discovery 5.1 (2010): 51-63.