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FRAP stands for ‘fluorescence recovery after photobleaching’. It is an optical technique that has been broadly adopted in the bioscience community to quantify two dimensional lateral diffusion rates of fluorescently labeled molecules in a thin film layer.
Lipid cubic phase (LCP) protein crystallization has proven successful to obtain high resolution structure of the GPCR protein. This method depends on the diffusion ability of the protein in a lipid matrix. Membrane protein diffusion rates are affected by the following parameters: protein aggregation state, the structural parameters of the LCP, and chemical environment. Screening crystallization conditions in this multi-parameter space is a research bottleneck. Automated high throughput LCP-FRAP assay is able to prescreen crystallization conditions and identify positive conditions.
FRAP is offered as a standalone system but can also be integrated with the ROCK IMAGER 1000. The ROCK IMAGER 1000 with FRAP maintains two independent imaging stations. You are able to run a FRAP experiment in parallel with bright field color imaging.
You can opt to run FRAP in a single LCP drop. However. the system is built with a hands-free operation in mind. You only need to set up the FRAP inspection schedule; the system will automatically load the plate and finish the whole FRAP inspection.
Protein mobility results are saved in a central database and presented to the user in a canvas view. Color codes are assigned to each drop to highlight the positive drops.
Yes. Proteins must be labeled with a fluorescent dye. We suggest Cy3 as the preferred labeling dye.
All glass base plates and glass cover slips work very well. However, to achieve accurate and repeatable mobility measurements, we suggest 60 µm spacers to maintain a thin sample thickness. FRAP is measuring a two dimensional molecule diffusion rate. Thin sample thickness will eliminate the diffusion effects in the third dimension. Thin sample thickness will also facilitate high contrast bleaching spot, which leads to more accurate diffusion results.
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