Sep 30, 2023

HPLC Analysis of Nucleotides

DOI
https://dx.doi.org/10.17504/protocols.io.36wgq36p3lk5/v1
  • 1University of California, Berkeley;
  • 2Aligning Science Across Parkinson's
Annan SI Cook
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgq36p3lk5/v1
Protocol Citation: Xuefeng Ren, Annan Cook 2023. HPLC Analysis of Nucleotides. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq36p3lk5/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 24, 2023
Last Modified: May 31, 2024
Protocol  Integer ID: 88518
Keywords: ASAPCRN, phase hplc analysis of the nucleotide, hplc analysis of nucleotides ion, reversed phase hplc analysis, hplc analysis, nucleotides ion, nucleotide, pi3kc3
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000350
Abstract
Ion-pair reversed phase HPLC analysis of the nucleotide bound to PI3KC3-C1.
Attachments
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852-2201.pdf
53KB
Materials
Materials

  • PI3KC3-C1 sample
  • Heat block
  • Microcentrifuge
  • Tubes
  • 10 μM ATP, ADP, GTP, GDP standards
  • HPLC system with UV detector
  • C18 reverse-phase HPLC column
  • Mobile phase Buffer A: 100mM KH2PO4, 5mM tetrabutylammonium bromide (TBA-B), pH 6.0, 1% acetonitrile (ACN)
  • Mobile phase Buffer B: 100mM KH2PO4, 5mM TBA-B, pH 6.0, 30% ACN
  • Gradient elution program (Chromeleon)
  • Wavelength set to 254 nm
  • Pipettes and tips
Denaturation of PI3KC3-C1
10m
Heat the PI3KC3-C1 sample in a heat block at 90 °C for 00:10:00 to denature the protein.
10m
Centrifugation
15m
After denaturation, centrifuge the sample at 21000 rpm, 00:15:00 to pellet any precipitated PI3KC3-C1.
15m
Supernatant Transfer
Carefully transfer the supernatant containing the released nucleotide to a clean tube, leaving behind the pellet.
Preparation of Nucleotide Standards
Prepare 10 micromolar (µM) stock solutions of ATP, ADP, GTP, and GDP standards in 25 millimolar (mM) HEPES 7.5 , 150 millimolar (mM) NaCl, 2 millimolar (mM) MgCl2, and 2 millimolar (mM) TCEP.
HPLC Column Equilibration
Equilibrate the C18 reverse-phase HPLC column with the mobile phase Buffer A ( and Buffer B according to the manufacturer's instructions.
HPLC Gradient Program
30m
Set up the HPLC system with the following parameters:

  • Mobile phase: gradient of Buffer A to Buffer B.
  • Gradient duration: 00:30:00 per run.
  • Wavelength for detection: 254 nm.
30m
Sample Injection
Inject 60 µL each of the nucleotide standards and the Protein nucleotide into the HPLC system.
Data Collection
Allow the HPLC system to run the samples through the column.
Record the retention times for each eluted nucleotide as they appear in the chromatogram.
Analysis
Compare the retention times of the eluted nucleotides in the sample to those of the known standards (ATP, ADP, GTP, GDP).
Identify the bound nucleotide(s) based on their retention times in the chromatogram.