How to Conduct a FRET Assay

Fluorescence resonance energy transfer (FRET) is an assay technique used to measure the presence and amount of binding between two reporter-linked proteins. This assay is commonly used to analyze potentially binding proteins in various types of cells, and to measure the effectiveness of conjugation-type enzymes. The detection equipment needed depends largely on the kinds of reporter molecules being used, though many times a simple 96-well plate reader can be used.

Things You'll Need

• Protein-of-interest/Reporter molecule constructs for each protein being tested
• 96-well plate block
• 96-well plate reader
• Reaction buffer (generally Tris- or Phosphate-buffered-saline-based)
• Conjugation enzyme (if necessary)

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Instructions

Setup and Reading

• 1

  Generate or prepare fusion constructs of each protein of interest, with each being linked to a fluorescence-transmission molecule such as green fluorescent protein (GFP), rhodamine or boron-dipyrromethene (BODIPY).

• 2

  Determine absorbance and emission wavelengths of each fluorescent component and of the overall assay. (For example, if the assay measures GFP-->Rhodamine fluorescence energy transfer, the input absorbance wavelength is that of GFP's absorbance (488 nm), and the emission detection wavelength is that for rhodamine (595 nm)).

• 3

  Create appropriate reaction buffer for the experiment, depending on the proteins of interest's biochemical properties. A TRIS-based buffer is often used, including calcium chloride and 0.05 percent Tween-20. Specific concentrations and components can be determined by searching for journal entries detailing similar reactions and conditions.

• 4

  Mix the two reporter-conjugated proteins of interest in various concentrations in the reaction buffer, and aliquot 50-200 ul per well. Add a conjugation enzyme (for example,sortase) to each sample well if appropriate. If performing an end-point assay, allow incubation at room temperature (or enzyme's optimal temperature) for a determined period of time, then read on 96-well plate reader. If performing a kinetics assay, proceed directly to reading (see below).

• 5

  Insert sample plate into 96-well plate reader. Create a new experiment page and access the settings menu. Input the correct absorbance (excitation) and emission wavelengths, then select the appropriate wells to be read.

  If performing a kinetics experiment, set the time-course of the experiment and the readings interval period (such as once every 10 minutes).

  Begin reading sample.

  Analyze data using Microsoft Excel's graphing function (or another graphing program such as GraphPad).