Membrane Protein Detergent Screening

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

Watch the video:

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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.

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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.