Sep 30, 2023

Public workspaceHPLC Analysis of Nucleotides

This protocol is a draft, published without a DOI.
  • 1University of California, Berkeley;
  • 2Aligning Science Across Parkinson's
Annan SI Cook
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Protocol Citation: Xuefeng Ren, Annan SI Cook 2023. HPLC Analysis of Nucleotides. protocols.io https://protocols.io/view/hplc-analysis-of-nucleotides-c2peydje
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 25, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 88518
Keywords: ASAPCRN
Funders Acknowledgement:
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
Denaturation of PI3KC3-C1
10m
10m
Heat the PI3KC3-C1 sample in a heat block at Temperature90 °C for Duration00:10:00 to denature the protein.
10m
Temperature
Centrifugation
Centrifugation
15m
15m
After denaturation, centrifuge the sample at Centrifigation21000 rpm, 00:15:00 to pellet any precipitated PI3KC3-C1.
15m
Centrifigation
Supernatant Transfer
Supernatant Transfer
Carefully transfer the supernatant containing the released nucleotide to a clean tube, leaving behind the pellet.
Preparation of Nucleotide Standards
Preparation of Nucleotide Standards
Prepare Concentration10 micromolar (µM) stock solutions of ATP, ADP, GTP, and GDP standards in Concentration25 millimolar (mM) HEPES Ph7.5 , Concentration150 millimolar (mM) NaCl, Concentration2 millimolar (mM) MgCl2, and Concentration2 millimolar (mM) TCEP.
HPLC Column Equilibration
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
HPLC Gradient Program
30m
30m
Set up the HPLC system with the following parameters:

  • Mobile phase: gradient of Buffer A to Buffer B.
  • Gradient duration: Duration00:30:00 per run.
  • Wavelength for detection: 254 nm.
30m
Sample Injection
Sample Injection
Inject Amount60 µL each of the nucleotide standards and the Protein nucleotide into the HPLC system.
Data Collection
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
Analysis
Compare the retention times of the eluted nucleotides in the sample to those of the known standards (ATP, ADP, GTP, GDP).
Analyze
Identify the bound nucleotide(s) based on their retention times in the chromatogram.