Encyclopedia of Experiments: Biological Techniques
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Transcript
Mix 3 nanomoles of the SBDS-FlAsH protein with 3 nanomoles of the Lumio Green dye in a 5-microliter volume of anisotropy buffer. Let the reaction proceed for 8 hours at 4 degrees Celsius. After 8 hours, dialyze the sample against the anisotropy buffer overnight to remove the free dye. Measure the absorbance at 280 nanometers and 508 nanometers in a spectrophotometer using a quartz cuvette. Then, use the Lambert-Beer law to quantify the percent of labeled protein as described in the text protocol.
Before measuring the anisotropy value, each titration step of the protein-ligand should be done carefully, ensuring that the entire sample is dispensed into the solution, and becomes homogeneous.
In a fluorescence cuvette, place 200 microliters of 30 nanomolar SBDS-FlAsH in anisotropy buffer, and titrate 2 microliters of 30 micromolar EFL1. Mix thoroughly, and let the reaction stand for 3 minutes before measuring the anisotropy and fluorescence value. Repeat this process until a total volume of 40 microliters of EFL1 has been added. As a final step, fit the data to a presumed binding model, as described in the text protocol.
