Unit

UNIT 1.16 Simultaneous Analysis of the Cyan, Green, and Yellow Fluorescent Proteins

  1. Andrew J. Beavis,
  2. Robert F. Kalejta

Published Online: 1 MAY 2001

DOI: 10.1002/0471142956.cy0116s16

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Beavis, A. J. and Kalejta, R. F. 2001. Simultaneous Analysis of the Cyan, Green, and Yellow Fluorescent Proteins. Current Protocols in Cytometry. 16:1.16:1.16.1–1.16.12.

Author Information

  1. Princeton University, Princeton, New Jersey

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: AUG 2001

Abstract

Flow cytometric analysis of fluorescent protein expressing cells is of particular interest to researchers in many areas. The detection of fluorescent proteins in cells allows one to monitor gene expression, determine intracellular protein localization, and identify transfected cells. Wild-type green fluorescent protein has limited utility as its spectral properties are not suitable for standard cytometers. Site-directed mutations have produced enhanced variants with improved extinction coefficient and quantum yield with standard 488-nm excitation. Other variants have been constructed with shifted excitation and emission maxima and high quantum yield. It is now possible to monitor multiple processes in a single cell and detect enhanced green, yellow, and cyan fluorescent proteins using a single excitation beam at 458 nm. The authors carefully describe the custom filter setup required to accomplish this and the Boolean gating logic for analysis of the various subpopulations expressing any given combination of fluorescent proteins.