title: Determining the number of specific proteins in cellular compartments by quantitative microscopy.
abstract: This protocol describes a method for determining both the average number and variance of proteins, in the few to tens of copies, in isolated cellular compartments such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number, but lack information on the variance, or they are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling of the cellular compartment with fluorescent primary-secondary antibody complexes, total internal reflection fluorescence microscopic imaging of the cellular compartment, digital image analysis and deconvolution of the fluorescence intensity data. A minimum of 2.5 d is required to complete the labeling, imaging and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes.
authors.affiliation: Department of Chemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Mutch
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Gadd
authors.forename: Bryant S
authors.initials: BS
authors.lastname: Fujimoto
authors.forename: Patricia
authors.initials: P
authors.lastname: Kensel-Hammes
authors.forename: Perry G
authors.initials: PG
authors.lastname: Schiro
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Bajjalieh
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Chiu
mesh_terms: D000465:Algorithms; D002451:Cell Compartmentation; D022083:Cytoplasmic Structures / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007091:Image Processing, Computer-Assisted / Q000379:methods; D044085:Microfluidics / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012984:Software
chemical_list: D011506:Proteins
references.citation: Trends Cell Biol. 1997 Dec;7(12):499-503
references.pmid: 17709014
references.citation: Science. 2005 Oct 14;310(5746):310-4
references.pmid: 16224022
references.citation: Prog Clin Biol Res. 1988;270:77-90
references.pmid: 3045836
references.citation: Neuron. 1989 Dec;3(6):715-20
references.pmid: 2577130
references.citation: J Am Chem Soc. 2006 Mar 15;128(10):3233-40
references.pmid: 16522104
references.citation: Biotechniques. 2005 Mar;38(3):429-46
references.pmid: 15786809
references.citation: Cell. 2006 Nov 17;127(4):831-46
references.pmid: 17110340
references.citation: Methods Cell Biol. 1989;30:245-70
references.pmid: 2648112
references.citation: EMBO J. 1988 Oct;7(10):3023-9
references.pmid: 2903047
references.citation: J Chromatogr B Biomed Sci Appl. 1999 Feb 5;722(1-2):89-102
references.pmid: 10068135
references.citation: Proteomics. 2008 Sep;8(18):3848-61
references.pmid: 18704904
references.citation: J Neurosci. 2011 Jan 26;31(4):1461-70
references.pmid: 21273430
references.citation: Nat Methods. 2005 Sep;2(9):677-84
references.pmid: 16118638
references.citation: Biophys J. 2007 Apr 15;92(8):2926-43
references.pmid: 17259276