Mar 27, 2025

Public workspacePrevention of Rhodanese Aggregation Assay

DOI
dx.doi.org/10.17504/protocols.io.j8nlkdbrxg5r/v1
  • Patricia Yuste-Checa1,
  • Andreas Bracher1,
  • F Ulrich Hartl1
  • 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Patricia Yuste-Checa
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkdbrxg5r/v1
Protocol Citation: Patricia Yuste-Checa, Andreas Bracher, F Ulrich Hartl 2025. Prevention of Rhodanese Aggregation Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkdbrxg5r/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: March 20, 2025
Last Modified: March 27, 2025
Protocol Integer ID: 124797
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol is based on Weber F, Hayer-Hartl M. Methods Mol Biol. (2000) and details how to efficiently monitor the prevention of rhodanese aggregation by the extracellular chaperone Clusterin using a spectrophotometer.
Materials
Buffers and reagents:
  • Rhodanese from bovine liver (Merck, R1756, MW 33,296 Da). Prepare a ~Concentration200 micromolar (µM) stock dilution in Concentration20 millimolar (mM) MOPS-NaOH pH Ph7.4 , Concentration50 millimolar (mM) NaCl, divide into small aliquots, flash-freeze in liquid nitrogen and store at Temperature-80 °C .
  • Denaturation buffer: Concentration6 Molarity (M) guanidinium-HCl, Concentration5 millimolar (mM) DTT (in water or PBS, depending on the reaction buffer).
  • PBS 1x
  • Clusterin (see protocol “Clusterin purification from HEK293E cells”, dx.doi.org/10.17504/protocols.io.bvvkn64w) or any other chaperone.




Prevention of Rhodanese Aggregation Assay
Prevention of Rhodanese Aggregation Assay
Thaw and lyophilize a rhodanese aliquot using a SpeedVac concentrator.
Resuspend lyophilized rhodanese with denaturation buffer to a final concentration of Concentration60 micromolar (µM) (D-Rhod).
Note
For example, a Amount20 µL aliquot at Amount8 µL pellet in Amount80.8 µL buffer. Amount1 µL of this stock will be used for each reaction.


Incubate at Temperature25 °C for Duration01:00:00 .

1h
Incubation
Make Clusterin stocks at Concentration30 micromolar (µM) and Concentration90 micromolar (µM) , for 1:1 and 1:3 D-Rhod:Clu molar ratios, respectively.

  • Prepare assay in a spectrophotometer microcuvette. Final volume Amount120 µL .
  • Use a spectrophotometer with temperature control of the cuvette compartment.
  • Equilibrate temperature at Temperature25 °C .
Add Amount2 µL Clusterin stock at Concentration30 micromolar (µM) or Concentration90 micromolar (µM) (final concentration 0.5 µM or 1.5 µM) to the bottom of the cuvette.

Pipetting
Add Amount117 µL PBS buffer and mix gently with the pipette. Equilibrate at Temperature25 °C in the cuvette compartment.
Note
NOTE: Other reaction buffers can be used to test the prevention of aggregation. For example, to test the prevention of aggregation of rhodanese by Clusterin at Ph5.2 use Concentration20 millimolar (mM) Na-acetate Ph5.2 , Concentration150 millimolar (mM) NaCl, Concentration2 millimolar (mM) CaCl2 buffer.

Pipetting
Add Amount1 µL of denatured rhodanese stock at Concentration60 micromolar (µM) (final concentration 0.5 µM) to the reaction mix.
Pipetting
Immediately mix by vortexing shortly, place the cuvette in the spectrophotometer, press autozero and start measurement at 320 nm wavelength every Duration00:00:02 .
Note
NOTE: Rhodanese rapidly aggregates once diluted in buffer. Clusterin at molar ratio D Rhod:Clu 1:3 almost completely prevents rhodanese aggregation.





Protocol references
Weber F, Hayer-Hartl M. Prevention of rhodanese aggregation by the chaperonin GroEL. Methods Mol Biol. 2000;140:111-5. doi: 10.1385/1-59259-061-6:111. PMID: 11484477.