Spherical Supported Lipid Bilayer Assay: A Technique to Observe the Septin Assembly on Microspheres
Transcript
First, vortex the bottle containing silica microspheres for 15 seconds. Then, bath-sonicate for one minute, and vortex again for 15 seconds to break any clusters. Mix the beads with SLBB and 10 microliters of 5-millimolar SUVs in a low-adhesion microcentrifuge tube.
Incubate the bead-lipid mixture for one hour at room temperature, on a shaker to prevent sedimentation. In the meantime, thaw a PEGylated coverslip, and glue a cut 0.2-milliliter PCR tube to it. After incubation, centrifuge the beads for 30 seconds at the designated RCF.
After the first spin, remove 50 microliters of supernatant. Then, add 200 microliters of PRB and mix by vigorous pipetting. For the next rounds, remove 200 microliters of PRB, and add another 200 microliters after the second and third spin, and add 220 microliters of PRB after the fourth spin.
If doing a competition assay with multiple bead sizes, prepare the reaction by mixing equal volumes of each bead size, and diluting 29 microliters of this bead mixture with 721 microliters of reaction buffer. Then, add 75 microliters of diluted beads and 25 microliters of the protein diluted in SSB to the wells and mix.
If measuring septins at a steady state, incubate the mixture at room temperature for one hour, and then, image by either near-TIRF or confocal microscopy.
