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J.C. Latham, R.A. Stein, D.J. Bornhop, H.S. Mchaourab; Label–Free Interferometric Detection of a–Crystallin Substrate Binding in a Microfluidic Device . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2007.
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© ARVO (1962-2015); The Authors (2016-present)
We report a new approach for the detection of protein–protein interactions between small heat shock proteins (sHSP) and their substrates. Using a chip–scale interferometer (CSI), analyses of binding are performed both in very small volumes (ca. nano– to picoliters) and without the use of any labeling moeity (fluorescent, radioactive, etc.).
The CSI consists of a coherent light source, a microfluidic chip with a detection volume on the order of 500pL and a phototrasnducer. Changes in the optical pathlength are manifested as phase changes of the backscatter interference patterns. The phase changes are detected using a linear CCD array, processed and displayed as a function of time. As a proof of concept, we used destabilized mutants of T4 Lysozyme (T4L) as substrates. T4L/sHSP binding experiments were conducted at various temperatures in microfluidic channels molded in poly(dimethylsiloxane) with an in–line mixer incorporated.
The homogeneous binding of αB–crystallin and Hsp27 to T4L is monitored in real–time, label–free. The data can be analyzed to determine ON and OFF rates or can represented as binding isotherms. Using CSI, we were able to reproduce the trends previosuly observed by fluorescence detection. αB–crystallin exhibited a higher affinity to the more destablized mutants of T4L. Both Hsp27 and αB–crystallin transition to two mode binding in repsonse to phosphorylation mimicking substitutions. Unlike detection by bimane fluorescence, CSI binding isotherms of Hsp27 were clearly biphasic presumably reflecting the different sizes of the complexes formed by high and low affinity binding.
CSI provides several advantages over other approaches for detection of chaperone/substrate interactions. By eliminating the need for probe attachment, it will allow the investigation of interactions with in–vivo substrates that are not amenable to high yield expression and labeling. The small volume of the measurement is conducive to analysis of multi–protein component solutions paving the way for high throughput analysis strategies using microfluidic chip technologies.
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