Sep 30, 2023

Public workspaceADP Glo Protocol

This protocol is a draft, published without a DOI.
  • 1University of California, Berkeley;
  • 2Aligning Sciences Across Parkinson's
Annan SI Cook
Open access
Protocol Citation: Annan SI Cook, Xuefeng Ren 2023. ADP Glo Protocol. protocols.io https://protocols.io/view/adp-glo-protocol-c2n6ydhe
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: 88510
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000350
Abstract
Protocol for ADP Glo (Promega) assay to measure the catalytic activity of the lipid kinase VPS34 on small unilammelar vesicles.
Attachments
852-2202.pdf
852-2202.pdf
34KB
Materials
Materials

  • Small unilamellar vesicles (SUVs) composed of
A
40% dioleoyl phosphatidylcholine (DOPC)
20% dioleoyl phosphatidylethanolamine
20% dioleoyl phosphatidylserine
20% liver phosphatidylinositol
All lipids from Avanti Polar Lipids.

  • HEPES pH 7.5
  • NaCl
  • MgCl2
  • MnCl2
  • TCEP
  • ATP
  • PI3KC3-C1
  • ADP-Glo ATP Depletion Reagent (Promega)

Equipment

  • Glass tube
  • N2 stream source
  • Vacuum dessicator
  • Benchtop centrifuge
  • Sonicator with a small probe tip
  • Water bath at 37°C
  • Liquid nitrogen
Preparation of Small Unilamellar Vesicles (SUVs)
Preparation of Small Unilamellar Vesicles (SUVs)
15m
Dehydrate Amount0.4 mg of the lipid mixture (40% DOPC, 20% DOPE, 20% DOPS, 20% liver phosphatidylinositol, Avanti Polar Lipids) in a glass tube using a gentle N2 stream.
Allow the lipids to dry DurationOvernight in a vacuum desiccator to remove excess chloroform.
Overnight
Rehydrate the dried lipids by adding Concentration25 millimolar (mM) HEPES Ph7.5 and Concentration200 millimolar (mM) NaCl.
Pipetting
Vortex the mixture to resolubilize the lipids.
Mix
Perform 10 cycles of freeze-thaw by immersing the lipids in liquid nitrogen for a few seconds and then in a Temperature37 °C water bath.
Sonicate the lipids using a small probe tip sonicator at 50% power in 2-second on/off cycles for a total of Duration00:15:00 TemperatureOn ice and a water mixture.
15m
Preparation of Buffers
Preparation of Buffers
Prepare a kinase dilution buffer.

Kinase dilution buffer
AB
HEPES pH 7.525 mM
NaCl200 mM
MgCl210 mM
MnCl21 mM
TCEP2 mM

Prepare a 2x lipid dilution buffer.
2x lipid dilution buffer
AB
HEPES pH 7.525 mM
NaCl200 mM
MgCl220 mM
MnCl22 mM
TCEP4 mM

ADP-Glo assay
ADP-Glo assay
1h 40m
In a reaction tube, combine the following components:
AB
50 nM PI3KC3-C19 μL
0.1 mg/mL SUVs in the 2x lipid dilution buffer9 μL
500 μM ATP in the same buffer2 μL

Mix the components gently.
Mix
Allow the reaction to proceed for Duration01:00:00 at TemperatureRoom temperature .
1h
Incubation
After the incubation, add Amount20 µL of the ADP-Glo ATP depletion reagent to each reaction tube.
Pipetting
Incubate the reaction tubes with the ADP-Glo ATP depletion reagent for Duration00:40:00 at TemperatureRoom temperature .
40m
Incubation
Measure the luminescent output for each condition tested using a luminometer or a plate reader.