Book chapter
A Guide to Tracking Single Transmembrane Proteins in Supported Lipid Bilayers
Lipid-Protein Interactions, pp.233-252
Methods in Molecular Biology, Humana Press
12/17/2012
Handle:
https://hdl.handle.net/2376/112345
PMID: 23404279
Abstract
The purpose of this chapter is to serve as a guide for those who wish to carry out experiments tracking single transmembrane proteins in planar supported membrane biomimetics. This chapter describes, in detail, the construction of a simple single-molecule microscope, which includes (1) a parts list, (2) an alignment procedure, (3) a calibration procedure, and (4) a procedure for measuring the mechanical stability of the instrument. It also gives procedures for making planar supported POPC bilayers on hydrophilically treated borosilicate and quartz, POPC/PEG-PE cushioned bilayers on hydrophilically treated surfaces, and POPC/PEG-PE cushioned bilayers on BSA passivated substrates. The procedure for the detergent-mediated incorporation of the transmembrane protein 5HT3A (a serotonin receptor) is also described and can be used as a starting point for other large non-self-inserting transmembrane proteins. The final experimental section of this chapter details different procedures for data analysis including (1) a quantitative analysis of mean displacements from individually tracked particles, (2) a Gaussian analysis of step-size distributions, (3) the Gaussian analysis of diffusion coefficients from ensembles of transmembrane proteins, and (4) a perspective associated with the interpretation of single-particle tracking data.
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Details
- Title
- A Guide to Tracking Single Transmembrane Proteins in Supported Lipid Bilayers
- Creators
- Kumud Raj Poudel - Department of Chemistry, Washington State University, Pullman, USAJeffrey P Jones - Department of Chemistry, Washington State University, Pullman, USAJames A Brozik - Department of Chemistry, Washington State University, Pullman, USA
- Publication Details
- Lipid-Protein Interactions, pp.233-252
- Academic Unit
- Chemistry, Department of
- Series
- Methods in Molecular Biology
- Publisher
- Humana Press; Totowa, NJ
- Identifiers
- 99900547635801842
- Language
- English
- Resource Type
- Book chapter