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Membrane Protein Detergent Screening

This tutorial focuses on detergent screening for membrane proteins using Fida measurements.

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Key Concepts in Fida Measurements

  • Fida Measurement Principles: In Fida measurements, the software fits raw data to determine diffusivity (indicated as "D") from the peak width using Stock Einstein's equation.
  • Importance residence time (tr): the time it takes for the peak top to reach the detector and is proportional to viscosity. Fida measurements directly measure viscosity, which is integral to the Stock Einstein equation.

Fidabio's Method for Detergent Screening is a rapid and sensitive method: determine the best detergent for solubilizing GFP membrane proteins in crude media without requiring purification.

Key Parameters Measured:

  • Hydrodynamic radius and peak area, with peak area proportional to fluorescent light.
  • Polydispersity index included in each measurement, eliminating the need for additional experiments.

Steps in Detergent Screening

Sample Preparation:

  1. Lysing cells, spinning down insoluble fractions, and resuspending in PBS.
  2. Performing a protein assay and diluting the protein to a specific concentration.

Solubilisation Process:

  1. Mixing lysate with PBS buffer and detergent, followed by a two-hour solubilisation process at 4°C on a tilting table.
  2. Ultracentrifugation step for further purification.

Fida Sample Preparation:

  1. Preparing two vials for each detergent: one with the protein in the desired detergent, the other with just PBS buffer and detergent.
  2. Setting up the Fida sequence for the experiment.

Experiment Execution:

  1. Testing different detergents and analysing data.
  2. Keeping crude membranes stable by using fresh samples and maintaining low temperatures during the run.

Data Analysis:

  1. Checking raw data and fitting data manually or using auto mode for multiple samples.
  2. Ranking detergents based on parameters like size and peak area.

Result Interpretation:

  1. Comparing peak intensities to determine the effectiveness of different detergents.
  2. Using the saved data for detailed analysis in software like Excel.

Considerations for Successful Screening

  • Sample Stability: Ensure that the solubilised membranes are stable by keeping them on ice and adding protease inhibitors.
  • Instrument Setup: Adjust the Fida instrument settings to maintain the sample at the desired temperature throughout the experiment.
  • Capillary Considerations: Decide whether to use standard or coated capillaries based on the presence of tailing effects in the data.