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Assaying for Inorganic Polyphosphate in Bacteria
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Protocol to quantify polyphosphate in human cell lines using a tagged PPBD peptide.

Javier Jiménez1, Blanca Lázaro1, Ana Sarrias1

  • 1Basic Sciences Department, Universitat International de Catalunya, 08195 Barcelona, Spain.

STAR Protocols
|May 11, 2022
PubMed
Summary

Researchers developed a new method to detect and quantify polyphosphate (polyP) in human cells. This technique utilizes a novel Xpress-tagged polyphosphate binding domain (PPBD) for improved visualization and measurement of nuclear polyP.

Keywords:
AntibodyCell BiologyMicroscopyProtein BiochemistryProtein expression and purification

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Area of Science:

  • Cell Biology
  • Biochemistry

Background:

  • Polyphosphate (polyP) is an evolutionarily conserved phosphate polymer.
  • Studying polyP in human cells is challenging due to its low concentration and instability.
  • Existing methods for polyP detection are limited.

Purpose of the Study:

  • To develop and validate a novel method for detecting and quantifying nuclear polyphosphate (polyP) in human cells.
  • To establish a reliable protocol for visualizing and measuring polyP using a specifically engineered binding domain.

Main Methods:

  • Expression and purification of Xpress-tagged polyphosphate binding domain (PPBD).
  • Detection and quantification of nuclear polyP in HEK293T cells using Xpress-PPBD.
  • Utilized Xpress antibody and Alexa-conjugated secondary antibodies for visualization.
  • Validated the protocol in SH-SY5Y, HeLa, and HEK293 cell lines.

Main Results:

  • Successfully expressed and purified Xpress-tagged PPBD.
  • Developed a robust protocol for detecting and quantifying nuclear polyP in various human cell lines.
  • The method allows for improved visualization and measurement of polyP compared to previous techniques.

Conclusions:

  • The developed Xpress-PPBD method provides a reliable approach for studying nuclear polyphosphate.
  • This protocol facilitates research into the biological roles of polyP in human cells.
  • The method is applicable across multiple human cell types, enhancing its utility.