Preparation of homogeneous samples of double-labelled protein suitable for single-molecule FRET measurements

E. Lerner, G. Hilzenrat, D. Amir, E. Tauber, Y. Garini, E. Haas

Research output: Contribution to journalArticlepeer-review

11 Scopus citations


Preparation of pure and homogenous site specifically single- and double-labelled biopolymers suitable for spectroscopic determination of structural characteristics is a major current challenge in biopolymers chemistry. In particular, proper analysis of single-molecule Förster resonance energy transfer measurements is based on the spectral characteristics of the probes. Heterogeneity of any of the probes may introduce errors in the analysis, and hence, care must be taken to avoid preparation of inhomogeneous labelled biopolymer samples. When we prepared samples of Escherichia coli adenylate kinase (AK) mutants labelled with either Atto 488 or Atto 647N, the products were spectrally inhomogeneous and the composition of the mixture changed gradually over time. We show here that the inhomogeneity was not a result of variation in the dye interaction with neighbouring side chains. Rather, the slow drift of the spectral characteristics of the probes was a characteristic of an irreversible chemical transformation probably due to the hydrolysis of the succinimide ring of the attached dye into its succinamic acid form. Overnight incubation of the labelled protein in mild basic solution accelerated the interconversion, yielding homogeneous labelled samples. Using this procedure, we obtained stable homogenous AK mutant labelled at residues 142 and 188.

Original languageEnglish
Pages (from-to)5983-5991
Number of pages9
JournalAnalytical and Bioanalytical Chemistry
Issue number18
StatePublished - Jul 2013

Bibliographical note

Funding Information:
Acknowledgments The help of Jordan Chill and Hugo Gotlib in the NMR analyses and of Rachel Persky and Bruria Shmerling in the mass spectral analysis is gratefully acknowledged. This study was supported by grants from the Israel Science Foundation (1102/06), the United States–Israel Binational Science Foundation (2005270), the EU Marie Curie TOK grant (29936) and by the Damadian Center for Magnetic Resonance Research, Bar-Ilan University.


  • Fluorescence
  • Labelling
  • Purification
  • Single-molecule FRET


Dive into the research topics of 'Preparation of homogeneous samples of double-labelled protein suitable for single-molecule FRET measurements'. Together they form a unique fingerprint.

Cite this