Single molecule assays of GFP-fused WT or mutant Kif21a constructs were performed in motility chambers as previously described (Qiu et al., 2012 (link)). Coverslips were sequentially coated with 1 mg/ml biotin-BSA and 0.5 mg/ml streptavidin to immobilize Taxol-stabilized Cy5-microtubules. Clarified cell lysates containing equal amounts of protein were diluted in motility buffer and added to the chamber. Images were recorded every 2s for 5 or 10min using an Olympus IX-81 TIRF microscope.
Ix 81 tirf microscope
Manufactured by Olympus
Sourced in Japan
The IX-81 TIRF microscope is a high-performance imaging system designed for Total Internal Reflection Fluorescence (TIRF) microscopy. It is capable of generating an evanescent field to selectively excite fluorophores near the coverslip surface, enabling high-resolution imaging of cell-substrate interactions and other surface-related phenomena.
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Lab products found in correlation
2 protocols using ix 81 tirf microscope
1
Single Molecule Kif21a Motility Assay
2
Plasma Membrane Translocation Assay
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To conduct the plasma membrane-translocation assay of the channel using TIRF imaging, HEK293 cells were plated at 35-mm coverslip bottom dish. Cell were transfected with M3, TRPC1α and YFP tagged TRPC4β or TRPC5 using FuGENE 6 (Promega) transfection reagent according to manufacturer’s protocol. Imaging was performed at room temperature with a 100x oil objective on the stage of an IX81 TIRF microscope (Olympus, Tokyo, Japan). Fluorescence images of YFP were taken using a diode laser at 488 nm.
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