A SEC profile is a powerful way to analyze the quality of a protein. The goal is to collect a SEC profile for a membrane protein using fluorescence, which can inform on sample quality. FSEC uses small quantities of sample and can be carried out for purification, is relatively simple, and can be carried out on equipment available in many labs.
Demonstrating the procedure will be Jack Wright, a Senior Protein Scientist from Peak Proteins. Begin by preparation of the cell palettes by thawing the cell palette stored at minus 80 degrees Celsius at room temperature for 15 minutes, or until the sample is no longer frozen. Move the sample immediately to ice.
To re-suspend and solubilize the sample, add two milliliters of the solubilization buffer to the cell palette. Incubate with end-over-end inversion at four degrees Celsius for 15 to 30 minutes. Then add pre-mixed detergent stock to make a final concentration of 1%dodecyl maltoside, or DDM, and 0.1%cholesterol hemisuccinate, or CHS.
Solubilize for 30 minutes with end-over-end inversion at four degrees Celsius. To perform a low-speed centrifugation step, centrifuge the sample in a benchtop centrifuge using a swing-out bucket and centrifuge at 2000g for 15 minutes. Then perform high-speed centrifugation by transferring the supernatant from the low-speed centrifugation to ultra-centrifugation tubes using a blunt-ended needle attached to a five-milliliter syringe.
Ensure not to disturb the palette. After balancing the pairs of tubes, place them in a fixed-angle ultra-centrifugation rotor and centrifuge at four degrees Celsius for 30 minutes at 250, 000g. Prepare the fast protein liquid chromatography, or FPLC system by filling the system with size-exclusion chromatography, or SEC buffer, and purging the pumps of air.
Connect the SEC column to the FPLC, ensuring no air enters the column. Pre-equilibrate the SEC column by washing it in 1.5 times column volumes of laboratory-grade distilled and filtered water, followed by 1.5 times column volumes of SEC buffer at the recommended flow rate and pressure for the column. To run the SEC experiment, transfer the supernatant from the high-speed centrifugation step to a one-milliliter syringe using a blunt-ended needle attached to the syringe.
Set the sample loop to load. Overfill a 500-microliter sample loop by injecting 600 to 700 microliters of the sample from the syringe into the loading port. Next, inject the sample from the loop into the column by emptying it with four milliliters of SEC buffer at the recommended flow rate and pressure.
Run the column at the same flow rate until the passing of 1.5 times the buffer volume, and start collecting 90 fractions of 0.2 milliliters after passing of 0.25 times the column volume. Using a multichannel pipette, transfer 90 microliters of laboratory-grade distilled water from a reservoir to each well of an opaque, flat-bottom, 96-well plate. Then transfer the 10 microliters of the samples from a 96-well plate to the opaque, flat-bottom, 96-well plate, and place the well plate into a plate reader.
For the fluorescent-labeled GFP, et the excitation as close as possible to 488 nanometers and detect the fluorescent emission as close as possible to 507 nanometers before measuring the signal. The investigation of dynamic range and lower limits of enhanced green fluorescent protein, or EGFP detection for the plate reader, indicated that the plate reader had a lower detection limit of 30 nanograms and a dynamic range of up to 500 nanograms of EGFP-labeled protein per well before signal saturation. The conversion of the elution volumes to Kav and plotting it against log molecular weights allowed the estimation of the molecular weight of the G-protein-coupled receptors by interpolation of the standard curve.
The optimal membrane extraction conditions were explored by testing the DDM and lauryl maltose neopentyl glycol against detergent-free extraction with styrene maleic acid copolymer. The effect of ligand addition on the fluorescence size-exclusion chromatography profile was investigated by adding sphingosine-1-phosphate, or S1P, to the sample during solubilization. The sample solubilized in the presence of S1P showed a superior trace with reduced aggregation.
The investigation of the long-term stability of serotonin receptor 5HT2AR under different conditions indicated that the protein in styrene maleic acid lipid particles remained stable for longer, even at unfavorable temperatures. The time between the point of solubilization and the sample passing down the SEC column is time critical. There must be no pauses, and the sample must be kept cold.
Following FSEC, an understanding of the best construct, detergent, or buffer will be gained. This allows protein purification to be optimized and supports downstream biophysical and structural characterization.
