Jul 25, 2022
Surface Density Calculation
- 1Hurley Lab
Protocol Citation: Liv Jensen 2022. Surface Density Calculation. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb65nnlpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: May 03, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 61857
Keywords: Surface Density Calculation, fluorescence intensity, ASAPCRN
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Abstract
This protocol details Surface Density Calculation.
Attachments
416-900.pdf
55KB
Materials
Materials: -
- Multichannel confocal fluorescence images; fluorophores in solution and on GUV at defined densities
- FIJI
- Image segmentation and quantification script (Python)
Surface Density Calculation
Surface Density Calculation
Quantify the fluorescence of a serial dilution of membrane fluorophore and of proteintag fluorophore in 1% SDS-containing buffer (10 micromolar (µM) , 6 micromolar (µM) , 2 micromolar (µM) , 1 micromolar (µM) , 0.5 micromolar (µM) of each fluor).
Compare the slopes of a plot of fluorescence intensity vs. concentration for the membrane dye and the protein dye to account for differences in optical properties.
Quantify the fluorescence of a series of GUV preparations containing varying mole percentage of membrane fluorophore (0.01%, 0.05%, 0.1%, 0.5%, 1%), using a rectangular selection of an approximately horizontal segment of each GUV.
Calculate the surface density of membrane dye from known surface area of lipids (~0.7nm2 /lipid).
Calculate the slope of a plot of membrane dye surface density vs. fluorescence signal, accounting for intensity from both lipid bilayer leaflets:
Relate surface density of lipid to surface density of protein label by normalizing to signal from bulk solution (from step 2):