title: A guide to tracking single transmembrane proteins in supported lipid bilayers.
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 5HT(3A) (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.
authors: Department of Chemistry, Washington State University, Pullman, WA, USA.
authors: Kumud Raj
authors: KR
authors: Poudel
authors: Jeffrey P
authors: JP
authors: Jones
authors: James A
authors: JA
authors: Brozik
mesh_terms: D001671:Biochemistry / Q000379:methods*; D004058:Diffusion; D007834:Lasers; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D044406:Receptors, Serotonin, 5-HT3; C028694:1-palmitoyl-2-oleoylphosphatidylcholine