OUTLINE OF THE LAB

    1. Generate an E. coli strain expressing a HIS-tagged ZsYellow with a mutation of your choice.

    2. Purify HIS-ZsYellow and your mutant protein using a nickel column.

    3. Analyze the absorbance, emission, and excitation spectra of HIS-ZsYellow and your mutant protein.

    4. Calculate the molar absorption coefficient and quantum yield of both proteins.

    5. Analyze the effect of pH, urea, and/or temperature on the stability and fluorescence characteristics of both proteins.


Equipment

Pictures





OTHER LAB MATERIALS

Spectrophotometer protocol

This protocol is for basic use of the Shimadzu BioSpec-mini spectrophotometer. (pdf file)

Spectrofluorometer protocol

This protocol is for basic use of the Shimadzu RF-1501 fluorescence spectrophotometer. (pdf document)

Living Colors User Manual, Volume II (Clontech)

This is the manual from Clontech's Living Colors fluorophores. These fluorophores originated from the GFP homologs derived from reef corals by Matz et al. (see the citation below). ZsYellow is among the Living Colors reef coral fluorescent proteins. (pdf document)


PAPERS RELATED TO THE LAB

Matz, Mikhail V., Arkady F. Fradkov, Yulli A. Labas, Aleksandr P. Savitsky, Andrey G. Zaraisky, Mikhail L. Markelov, and Sergey A. Lukyanov, Nature Biotech. 17, 1999, 969-73. Fluorescent proteins from nonbioluminescent Anthozoa species.

  • Six proteins homologous to green fluorescent protein (GFP) from nonbioluminescent reef corals were cloned, and their fluorescence spectra were analyzed.

    • Remington, S. James, Rebekka M. Wachter, Daniel K. Yarbrough, Bruce Branchaud, D. C. Anderson, Karen Kallio, and Konstantin A. Lukyanov, Biochemistry. 44, 2005, 202-12. zFP538, a Yellow-Fluorescent Protein from Zoanthus, Contains a Novel Three-Ring Chromophore.

  • This paper describes crystal structures of both the ZsYellow fluorophore (referred to in this paper as zFP538) and a green-emitting mutant in which a single residue has been changed (K66M). The relation of the ZsYellow chromophore to the GFP and DsRed chromophores is discussed.

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