Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics
Transkript
To begin plasma cleaning of the slides, purge the plasma cleaner for 5 minutes with oxygen to remove air from the lines and chamber. Arrange dry cover glass slides into a ceramic cradle. While purging, stream an inert gas over the micro cover glass slides to remove dust and particulates.
Place the cradle at the back of the plasma chamber so that the coverslips are parallel with the long edge of the chamber. Run the plasma cleaner for 15 minutes with oxygen at maximum power.
For chamber preparation, cut off the cap just below the frosted part of a 0.2-milliliter PCR tube. Paint the rim of the PCR tube with UV-activated adhesive, avoiding the inside of the tube. Gently place the PCR tube glued down in the center of a plasma-cleaned coverslip, then, place the chamber under long-wavelength UV light for 5 to 7 minutes to cure the adhesive.
To form a bilayer, add the reagents to the well. Gently shake the chambers from side to side to disrupt the SUVs, and then, incubate at 37 degrees Celsius for 20 minutes.
After incubation, rinse the bilayer 6 times with 150 microliters of SLBB, by pipetting to wash away excess lipids. Then, wash the bilayer 6 times with 150 microliters of reaction buffer, before incubating with the septins.
On the last wash step, remove the reaction buffer, leaving 75 microliters in the well. Add 25 microliters of septins diluted in SSB, and image by TIRF microscopy.
