Microchip Electrophoresis Assay for Calmodulin Binding Proteins.

The calcium signaling protein calmodulin regulates numerous intracellular processes. We introduce a sensitive microchip assay to separate and detect calmodulin binding proteins. The assay utilizes an optimized microchip electrophoresis protein separation platform with laser-induced fluorescence detection. Fluorescence-labeled calmodulin modified with a photoreactive diazirine crosslinker allowed selective detection of calmodulin binding proteins. We demonstrate successful in-vitro crosslinking of calmodulin with two calmodulin binding proteins, calcineurin and nitric oxide synthase. We compare the efficacy of commonly applied electrophoretic separation modes: microchip capillary zone electrophoresis, microchip micellar electrokinetic chromatography/gel electrophoresis, and nanoparticle colloidal arrays. Out of the methods tested, polydymethylsiloxane/glass chips with microchip zone electrophoresis gave the poorest separation, whereas sieving methods in which electro-osmotic flow was suppressed gave the best separation of photoproducts of calmodulin conjugated with calmodulin binding proteins.

Comparison of separation modes for microchip electrophoresis of proteins.

Separation of a set of model proteins was tested on a microchip electrophoresis analytical platform capable of sample injection by two different electrokinetic mechanisms. A range of separation modes-microchip capillary zone electrophoresis, microchip micellar electrokinetic chromatography, and nanoparticle-based sieving-was tested on glass and polydimethylsiloxane/glass microchips and with silica-nanoparticle colloidal arrays. The model proteins calmodulin (18 kiloDalton), bovine serum albumin (66 kDa), and concanavalin (106 kDa) were labeled with Alexa Fluor 647 for laser-induced fluorescence detection. The best separation and resolution were obtained in a silica-nanoparticle colloidal array chip.