UNIT 14.14 Fluorescent Labeling of Specific Cysteine Residues Using CyMPL

  1. Michael C. Puljung,
  2. William N. Zagotta

Published Online: 1 NOV 2012

DOI: 10.1002/0471140864.ps1414s70

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Puljung, M. C. and Zagotta, W. N. 2012. Fluorescent Labeling of Specific Cysteine Residues Using CyMPL. Current Protocols in Protein Science. 70:14.14:14.14.1–14.14.10.

Author Information

  1. Department of Physiology and Biophysics, University of Washington, Seattle, Washington

Publication History

  1. Published Online: 1 NOV 2012
  2. Published Print: NOV 2012


The unique reactivity and relative rarity of cysteine among amino acids makes it a convenient target for the site-specific chemical modification of proteins. Commercially available fluorophores and modifiers react with cysteine through a variety of electrophilic functional groups. However, it can be difficult to achieve specific labeling of a particular cysteine residue in a protein containing multiple cysteines, in a mixture of proteins, or in a protein's native environment. This unit describes a procedure termed CyMPL (Cysteine Metal Protection and Labeling), which enables specific labeling by incorporating a cysteine of interest into a minimal binding site for group 12 metal ions (e.g., Cd2+ and Zn2+). These sites can be inserted into any region of known secondary structure in virtually any protein and cause minimal structural perturbation. Bound metal ions protect the cysteine from reaction while background cysteines are covalently blocked with non-fluorescent modifiers. The metal ions are subsequently removed and the deprotected cysteine is labeled specifically. Curr. Protoc. Protein Sci. 70:14.14.1-14.14.10. © 2012 by John Wiley & Sons, Inc.


  • fluorescence;
  • FRET;
  • metal binding;
  • cysteine modification