Essential metal uptake in gram-negative bacteria: X-ray fluorescence, radioisotopes, and cell fractionation

Christopher D. Radka, Lauren L. Radford, Adriana V.F. Massicano, Lawrence J. Delucas, Suzanne E. Lapi, Stephen G. Aller

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for the purpose of biophysical characterization, and measure substrate transfer between periplasmic and cytoplasmic compartments. By carefully limiting the time that the periplasm is separated from the cytoplasm, the experimenter can extract the protein of interest and assay each compartment individually for substrate without carry-over contamination between compartments. The extracted protein from fractionation can then be further analyzed for three-dimensional structure determination or substrate-binding profiles. Alternatively, this method can be performed after incubation with a radiotracer to determine total percent uptake, as well as distribution of the tracer (and hence metal transport) across different bacterial compartments. Experimentation with a radiotracer can help differentiate between a physiological substrate and artefactual substrate, such as those caused by mismetallation. X-ray fluorescence can be used to discover the presence or absence of metal incorporation in a sample, as well as measure changes that may occur in metal incorporation as a product of growth conditions, purification conditions, and/or crystallization conditions. X-ray fluorescence also provides a relative measure of abundance for each metal, which can be used to determine the best metal energy absorption peak to use for anomalous X-ray scattering data collection. Radiometal uptake can be used as a method to validate the physiological nature of a substrate detected by X-ray fluorescence, as well as support the discovery of novel substrates.

Original languageEnglish
Article numbere57169
JournalJournal of Visualized Experiments
Issue number132
StatePublished - Feb 1 2018

Bibliographical note

Publisher Copyright:
© 2018 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.


  • Chemistry
  • Fractionation
  • Issue 132
  • Periplasm
  • Plague
  • Radioactivity
  • Radiotracer
  • Substrate-Binding Protein (SBP)
  • Transition Metal
  • X-Ray Fluorescence
  • Yersinia pestis
  • YfeA

ASJC Scopus subject areas

  • General Neuroscience
  • General Chemical Engineering
  • General Biochemistry, Genetics and Molecular Biology
  • General Immunology and Microbiology


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