How to screen detergents for membrane protein stability?

Flow Induced Dispersion Analysis (FIDA) screens detergents for membrane proteins by measuring the hydrodynamic radius, peak area and polydispersity index of a GFP-tagged membrane protein solubilised in each detergent. It is a rapid and sensitive method to determine the best detergent for solubilising GFP membrane proteins in crude media, without requiring purification.

This tutorial focuses on detergent screening for membrane proteins using Fida measurements. Watch the video:

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 the Stokes-Einstein equation.
• Residence time (t_R): the time it takes for the peak top to reach the detector, which is proportional to viscosity. Fida measurements directly measure viscosity, which is integral to the Stokes-Einstein equation.

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

Which detector to use?

Use the 480 nm detector for GFP-tagged membrane proteins, and the 530 nm detector for mCherry-tagged membrane proteins. Both are used for membrane protein detergent screening.

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.

The PDI parameter is an important criterion for selecting solubilising detergents during membrane protein solubilisation. When you solubilise a membrane protein, a low PDI means you have uniform protein-detergent complexes: good solubilisation, likely resulting in monodisperse complexes, which are ideal for experiments like crystallography, cryo-EM or functional assays. A high PDI means you have heterogeneous complexes: some are small, some are large aggregates, or mixed oligomeric states, which suggests poor solubilisation, aggregation or instability. This is covered in the Polydispersity course in the Sample Characterization & QC section of the Fidabio Learning Hub.

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. See How to coat a capillary to prevent sample sticking? and How do you stop protein sticking to surfaces?

What results has FIDA given for detergent screening?

In the poster High Throughput detergent screening for membrane proteins using nanoliters of sample, led by the National Physical Laboratory (NPL) with Fida Biosystems, FIDA screened 11 detergents for their ability to solubilise a GFP-tagged membrane protein (GFP-LacY) directly in crude cell lysate, from 1.5 µL of sample. The FIDA detergent ranking matched fluorescence size-exclusion chromatography (FSEC), while screening time fell from about 20 hours to under 3.

Frequently asked questions

Does the membrane protein need to be purified before detergent screening?

No. FIDA screens GFP-tagged membrane proteins in crude media, without requiring purification.

Which parameters rank the detergents?

Size (hydrodynamic radius) and peak area, with the polydispersity index from the same measurement.

What to do if your detergent readout shows bubbles?

An air bubble in the indicator well leads to a non-Gaussian profile, as in the readout above, and the data cannot be analysed. Extra care must be taken while loading samples containing detergents. Air bubbles can be prevented by slow release of sample on the surface of the plate or vial. If possible, avoid loading small volumes. Centrifuging the plate before loading it onto the instrument may help release air bubbles. This is covered in the My data looks like... course in the Troubleshooting section of the Fidabio Learning Hub.

Related resources

Want to see more membrane protein work with FIDA? See the application notes High throughput detergent screening for membrane proteins and Functional Characterization of Nanodisc embedded GPCR, and the poster FIDA-Based Quantification of Small Molecule Binding to Membrane Proteins in Solution.

New to membrane protein work with FIDA? The membrane proteins page shows the application, and What is hydrodynamic radius? explains what hydrodynamic radius is and how it is measured. To find out whether FIDA fits your membrane protein work, book a discovery call.