Jul 11, 2019

Public workspaceATP synthase activity assay (radioactive)

Journal of Bacteriology
DOI
dx.doi.org/10.17504/protocols.io.48qgzvw
  • 1Heinrich-Heine Universität Düsseldorf;
  • 2Institute of Biology III, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany;
  • 3Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan;
  • 4Institute for Microbiology and Molecular Biology, Justus-Liebig University, 35392 Giessen, Germany;
  • 5College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan;
  • 6Institute for Synthetic Microbiology, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich Heine University Duesseldorf, 40225 Duesseldorf, Germany
  • Axmann Lab
  • CyanoWorld
Anika Wiegard
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DOI: dx.doi.org/10.17504/protocols.io.48qgzvw
External link: https://doi.org/10.1128/JB.00478-19
Protocol Citation: Anika Wiegard, Christin Köbler, Katsuaki Oyama, Anja K. Dörrich, Chihiro Azai, Kazuki Terauchi, Annegret Wilde, Ilka Maria IM Axmann 2019. ATP synthase activity assay (radioactive). protocols.io https://dx.doi.org/10.17504/protocols.io.48qgzvw
Manuscript citation:
Wiegard A, Köbler C, Oyama K, Dörrich AK, Azai C, Terauchi K, Wilde A, Axmann IM, Array. Journal of Bacteriology 202(4). doi: 10.1128/JB.00478-19
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: July 06, 2019
Last Modified: July 11, 2019
Protocol Integer ID: 25584
Keywords: ATP synthesis, thin layer chromatography, TLC, KaiC
Abstract
This protocol describes how to detect synthesis of [α32P]ATP from [α32P]ATP by recombinant KaiC proteins. Radioactive nucleotides are separated via thin layer chromatography using TLC PEI Cellulose F plates as stationary phase and LiCl as soluble phase. The principle of this method is based on Egli et al. (Egli M, Mori T, Pattanayek R, Xu Y, Qin X, Johnson CH.2012. Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism. Biochemistry 51:1547-58.)
Materials
MATERIALS
ReagentMagnesium chloride hexahydrateMerck MilliporeSigma (Sigma-Aldrich)
Reagent[y-32P]ATP (3000 Ci/mmol)Hartmann Analytic GmbHCatalog #SRP-301
ReagentTris(hydroxymethyl)aminomethaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #252859-500G
ReagentNaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014
ReagentEDTA
ReagentLithium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #793620
ReagentAdenosin-5-triphosphate disodium salt (ATP)Carl RothCatalog #HN35.1
ReagentTLC PEI Cellulose F plates Merck Millipore (EMD Millipore)Catalog #1055790001
ReagentAdenosine 5′-diphosphate sodium salt (ADP)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A2754-100MG
Reagent[alpha-P32]ADP 6000 Ci/mmol 10 mCi/mlHartmann Analytic GmbHCatalog #SRP-227
preparation
preparation
  • express and purify KaiC
  • optional: phosphorylate or dephosphorylate KaiC prior to analysis
incubation
incubation
  • prepare a 25 µl mastermix with 3 μM KaiC in ATP synthesis buffer (20 mM Tris/HCl (pH8), 150 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, 0.5 mM ATP)
  • add 2 ul [α-32P]ADP (= 20 μCi)
  • freeze one 10 µl aliquot at -20 °C (0h, -20 °C)
  • incubate one 10 µl aliquot for 2 hours at 30 °C
  • as a control, perform the same reaction but in the presence of 0.5 mM (non-radioactive) ADP
  • in total you will have 4 samples for every KaiC protein of interest:
sample0.5 mM (non-radioactive) ADPincubation for 2h at
control 0h+-20 °C
control 2h+30 °C
sample 0h--20 °C
sample 2h-30 °C

thin layer chromatography
thin layer chromatography
  • spin down briefly and dilute samples 1:20 in H2O
  • spot 0.5 μl of the diluted reaction mixtures and [α-32P]ADP and [γ-32P]ATP (as size controls) onto a TLC PEI Cellulose F plates (Merck Millipore) - place the spots approx 1-2 cm from the bottom of the plate, mark the level with a pencil
  • wait until spots are dried
  • pour a small amount of 1 M LiCl as mobile phase in a thin layer chromatography chamber (the solvent level must be lower than the distance of your spots from the bottom of the TLC plate)
  • close the lid and wait a few minutes
  • place the TLC plate in the chamber, close the lid and allow the mobile phase to be drawn up the plate (without reaching the end of the TLC plate)
  • remove and dry the TLC plate
detection and analysis
detection and analysis
  • expose dried plates to an autoradiography screen and detect signals using for example a Personal Molecular Imager FX system (Bio-Rad)
  • analyze the samples (for example with ImageLab software (Bio-Rad))
  • for each lane determine the signal intensity of all detectable spots
  • calculate the relative intensity of [α-32P]ATP as percentage of all signals in the corresponding lane
  • for normalization substract the relative [α-32P]ATP intensity of the -20 °C sample containing 0.5 mM ADP from the relative [α-32P]ATP intensity in the other samples