Spherical Supported Lipid Bilayer Assay: A Technique to Observe the Septin Assembly on Microspheres

Abstract

Source: Curtis, B. N. et al., Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions. J. Vis. Exp. (2022).

This video describes the technique of studying the septin protein assembly on microspheres coated with lipid bilayers. This technique helps in understanding the curvature-dependent assemblies of protein on curved surfaces.

Protocol

1. Spherical supported lipid bilayers

NOTE: This protocol uses silica microspheres suspended in ultrapure water at 10% density. For any work on the kinetic parameters of protein assembly, it is important to strictly control the total membrane surface area between experiments and curvatures. Table 1 shows the corrected volumes of beads and buffer to maintain 5 mm² of total membrane surface area.

1. Bilayer formation
   NOTE: As for planar bilayers, it is important to have high-quality SUVs for robust bilayer formation. Table 1 list....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 mL PCR Tubes with flat cap, Natural	Watson	137-211C(EX)
0.5 mL low adhesion tubes	USA Scientific	1405-2600
Beta mercaptoethanol (BME)	Sigma-Aldrich	M6250-100ML
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A4612-25G
Coverglass for making PEGylated coverslips	Thermo Scientific	152450	Richard-Allan Scientific SLIP-RITE Cover Glass 24x50 #1.5
DOPC	Avanti Polar Lipids	850375
Egg Liss Rhodamine PE	Avanti Polar Lipids	810146
HEPES	Sigma Aldrich	H3375-1KG
Methyl cellulose 4000cp	Sigma-Aldrich	M052-100G
Mini centrifuge
Non-Functionalized Silica Microspheres	Bangs Laboratories, Inc.	Depends on size: SS0200*-SS0500*	Silica in aqueous suspension
Optical Adhesive	Norland Thorlabs	NOA 68	Flexible adhesive for glass or plastics
Potassium chloride	VWR	0395-1kg
Sonicator bath	Branson	1510R-MT	Bransonic Ultrasonic cleaner. 50-60 Hz. Output: 70W
Soy PI	Avanti Polar Lipids	840044
Tabletop centrifuge	Eppendorf	22331
UV Lamp	Spectroline	ENF-260C	115 Volts, 60 Hz, 0.20 AMPS