Mar 19, 2025

Public workspaceClusterin-VLDLR-ed Binding Assay

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l26m2qv1y/v1
  • Andreas Bracher1,
  • Patricia Yuste-Checa1,
  • F Ulrich Hartl1
  • 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany
Patricia Yuste-Checa
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.4r3l26m2qv1y/v1
Protocol Citation: Andreas Bracher, Patricia Yuste-Checa, F Ulrich Hartl 2025. Clusterin-VLDLR-ed Binding Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l26m2qv1y/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 11, 2025
Last Modified: March 19, 2025
Protocol Integer ID: 124567
Keywords: ASAPCRN, interaction between clusterin, very low density lipoprotein receptor, domain of very low density lipoprotein receptor, clusterin, ed binding assay this protocol detail, ed binding assay, vldlr
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK

The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
This protocol details how to show the interaction between Clusterin (Clu) and the C-tagged ecto-domain of Very Low Density Lipoprotein Receptor (VLDLR) in vitro by immunoprecipitation.
Materials
  • C-tag wash buffer
AB
Tris-HCl pH 7.420 mM
NaCl100 mM
CaCl22 mM
  • C-tag elution buffer
AB
Tris-HCl pH 7.020 mM
MgCl22 M
CaCl22 mM
  • CaptureSelect C-tag affinity resin (Thermo Fisher Scientific, 2943072005)
  • Empty spin columns for standard 1.5 ml reaction tubes (Mo Bi Tec, M1002S)
  • Clusterin (see protocol “Clusterin purification from HEK293E cells”, dx.doi.org/10.17504/protocols.io.bvvkn64w).
  • RAP (see protocol “Purification of recombinant Low Density Lipoprotein Receptor Related Protein Associated Protein 1 (LRPAP1, RAP) from Escherichia coli”, dx.doi.org/10.17504/protocols.io.rm7vzxpb2gx1/v1)
  • VLDLR-ed (VLDLR(28-797)Δ(751-779)-C-tag, derived from Uniprot isoform sequence P98155-2) (see protocol “Very low-density lipoprotein receptor (VLDLR)-C-tag purification from HEK293E cells”, dx.doi.org/10.17504/protocols.io.j8nlkoqw1v5r/v1)
  • NuPAGE 4–12% Bis-Tris SDS gels (Thermo Fisher Scientific)
  • NuPAGE MES SDS running buffer (Thermo Fisher Scientific)
  • 2x SDS-PAGE sample buffer
AB
Tris-HCl pH 6.8100 mM
Dithiothreitol (DTT)0.2 M
Sodium dodecyl sulfate (SDS)4%
l-1 bromophenol blue2 g
Glycerol20%
Note
NOTE: Prepare by mixing 1.54 g DTT, 0.10 g bromophenol blue, 10 ml glycerol, 5 ml of 1 M Tris-HCl pH 6.8 stock and 20 ml of 10% (w/v) SDS stock. Top up with Milli-Q de-ionized water to 50 ml total volume. Make 1-ml aliquots and store at -20 °C.

  • Pre-stained molecular weight SDS-PAGE marker (PageRuler Prestained Protein Ladder, 10 to 180 kDa, ThermoFisher)
  • Select Western blot transfer stacks nitrocellulose (Invitrogen)
  • Tris-buffered saline containing 0.05% Tween 20 (0.05% TBS-T)
TBS
AB
Tris-HCl pH 7.510 mM
NaCl150 mM

Note
NOTE: Prepare freshly from 10x TBS-T stock, 100 mM Tris-HCl pH 7.5, 1500 mM NaCl, 0.5% Tween 20.

  • Stripping buffer
AB
Glycine pH 2.020 mM
SDS2%

  • PBS-T (1x PBS with 0.1% Tween 20)
  • 3% bovine serum albumin (BSA) (Merck) in TBS-T. Store at 4 °C.
  • Mouse monoclonal Clu-α antibody (Santa Cruz Biotechnology, sc-5289, 1/1000 dilution)
  • Conjugated goat-anti mouse immunoglobulin G (IgG)-horseradish peroxidase (HRP) (Merck, A4416)
  • CaptureSelect biotin anti-C-tag conjugate (Thermo Fisher Scientific, 7103252100, 1/2000 dilution) Streptavidin-HRP (Pierce, 21130, 1/10000 dilution)
  • Immobilon Forte Western HRP substrate (Merck)

Instruments
  • Microcentrifuge for 1.5 ml reaction tubes (Eppendorf)
  • Vortex mixer (Vortex-Genie 2, Scientific Industries)
  • Thermo mixer (Eppendorf Thermomixer Comfort)
  • Semi-dry blotting apparatus (Power Blotter XL, Invitrogen)
  • Luminescence gel documentation system (ImageQuant LAS 4000 mini, General Electric)

ReagentCaptureSelect™ C-tagXL Affinity MatrixThermo FisherCatalog #2943072005
ReagentClusterin-α Antibody (B-5)Santa Cruz BiotechnologyCatalog #sc-5289
ReagentAnti-Mouse IgG (whole molecule)–Peroxidase antibody produced in goatMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4416
ReagentCaptureSelect™ Biotin Anti-C-tag ConjugateThermo FisherCatalog #7103252100

Troubleshooting
Preparatory Work
Completely resuspend C-tag affinity resin in storage buffer by gentle shaking.
Transfer approximately Amount50 µL C-tag affinity resin into each empty spin columns.
Note
Consider the dilution factor in the suspension, e.g. if the total volume is twice the bead volume, transfer 100 µl suspension. The nozzle of the pipette tip might be too narrow for the gel beads. Cut off the tip with scissors for easy transfer.


Pipetting
Let the storage buffer pass through by spinning at Centrifigation2000 rpm, 00:02:00 in a microcentrifuge.

2m
Centrifigation
Equilibrate columns with Amount250 µL C-tag wash buffer.

Remove residual C-tag wash buffer by spinning at Centrifigation2000 rpm, 00:02:00 in a microcentrifuge.

2m
Centrifigation
Plug the column outlets.
Complex Formation and Column Binding
Use following concentrations (µM) for your samples:
ABCD
SampleCluVLDLR-edRAP
Clu-Bkg5
Clu-VLDLR55
RAP-VLDLR (RV)55
RAP-Bkg (R)5
Calculate the necessary volumes of the Clusterin, VLDLR-ed and RAP stock solutions for Amount100 µL total volume. The volume of C-tag affinity resin is Amount50 µL . Fill up to Amount100 µL total volume with C-tag wash buffer.

Resuspend C-tag affinity resin by gentle vortexing.
Remove from each column Amount10 µL sample (“Input”) for SDS-PAGE.

Incubate for Duration02:00:00 at Temperature25 °C with gentle shaking (Shaker750 rpm ) in thermomixer.

2h
Incubation
Remove column plugs. Place fresh 1.5 ml collection tubes under the columns. Label tubes.
Harvest unbound residual C-tag wash buffer by spinning at Centrifigation2000 rpm, 00:02:00 in a microcentrifuge.

2m
Centrifigation
Replace collection tubes. Label tubes.
Wash four times with Amount100 µL C-tag wash buffer. Each time resuspend resin, followed by centrifugation. Collect wash solution.

Wash
Replace collection tubes. Label tubes.
Elute bound proteins three times Amount50 µL C-tag elution buffer. Each time resuspend resin, followed by centrifugation. Collect eluate.
SDS-PAGE
  • Prepare SDS-PAGE samples by mixing sample and 2x SDS-PAGE sample buffer at 1:1 ratio.
  • Then heat SDS-PAGE samples to Temperature95 °C for Duration00:05:00 in metal heating block, followed by cooling in a cold metal block.
  • Centrifugation for Centrifigation14000 rpm, 00:01:00 in microcentrifuge.
Note
The DTT in the 2x SDS-PAGE sample buffer quickly perishes by air oxidation. Keep chilled on ice! To avoid Clusterin gel artifacts by incomplete reduction of its five disulfide bonds, do not re-use 2x SDS-PAGE sample buffer, once it was thawed.


6m
Centrifigation
Temperature
Assemble electrophoresis apparatus with NuPAGE 4-12% Bis-Tris SDS gels. Fill inner and outer chamber with NuPAGE MES SDS running buffer (1x).
Load NuPAGE 4-12% Bis-Tris SDS gels with input samples (Amount5 µL ), unbound (Amount5 µL ), wash (Amount20 µL ) and elution (Amount7.5 µL ) SDS-PAGE samples. Use pre-stained SDS-PAGE molecular weight marker (Amount5 µL ) as a standard.
Note
NOTE: The unequal loading volumes result in equal dilution on the gel.


Run electrophoresis for Duration00:45:00 at a constant voltage of 200 V.

45m
Analyze gel with RAP-containing samples by Coomassie-blue staining.
Note
This will show the outcome of the binding experiments with RAP as a positive control.

Fig. 1 Coomassie-stained SDS-PAGE showing the binding of RAP to immobilized C-tagged VLDLR-ed (VR). RAP alone (R) shows only residual binding to the C-tag affinity resin. The bands for VLDLR-ed appear fuzzy probably because of heterogeneity of attached O- and Nglycans.

Analyze
Western Blot Transfer and Anti-Clusterin Immunostaining
  • Carefully pry open the plastic casing of the NuPAGE 4–12% Bis-Tris SDS gel by using a so-called gel knife (Invitrogen) or similar.
  • Remove the loading pockets with a scalpel while the gel is still attached to the plastic.
Place the gel into a staining dish and briefly wet with de-ionized water.
For electro-blotting, follow the steps in the instructions to the Power Blotter system (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017051_PBSelectStacks_QR.pdf).
After disassembly of the blotting stack, attach the nitrocellulose membrane to the inner wall of a tubular vessel and wash three times with Amount10 mL TBS-T while tumbling on a roller mixer.
Note
The protein bands have to face inside. The complete surface of this membrane face has to be accessible to the buffer. Do not touch membrane with bare fingers.


Wash
Block nitrocellulose membrane by incubation with Amount10 mL 3% BSA in TBS-T for Duration01:00:00 at TemperatureRoom temperature using a tubular vessel and a roller mixer.

1h
Incubation
Wash the nitrocellulose membrane three times with Amount10 mL TBS-T.

Wash
Incubate with mouse monoclonal Clu-α antibody, 1/1000 diluted with Amount10 mL TBS-T, DurationOvernight in the cold room using a tubular vessel and a roller mixer.

Incubation
Overnight
Wash the nitrocellulose membrane three times with Amount10 mL TBS-T.

Wash
Incubate with conjugated goat-anti mouse IgG-HRP, 1/2000 diluted with Amount10 mL TBS-T for Duration01:00:00 at TemperatureRoom temperature using a tubular vessel and a roller mixer.

1h
Incubation
Wash the nitrocellulose membrane three times with Amount10 mL TBS-T.

Wash
Transfer the nitrocellulose membrane onto a glass plate.
Using the gel documentation system, take a photo in epi-illumination mode.
Cover the nitrocellulose membrane with Amount3 mL Immobilon Forte Western HRP substrate.
Note
Do not move the glass plate.

Record 10 s-incremental exposures in luminescence mode until the first pixels start to saturate.
Membrane Stripping and Anti-C-tag Immunodetection

Note
This is an optional control to make sure that nothing went wrong in the initial steps (steps 8-17).
Wash the nitrocellulose membrane three times with Amount10 mL water using a tubular vessel and a roller mixer.

Wash
To strip the antibodies from the nitrocellulose membrane,

Incubation
Incubate the nitrocellulose membrane twice with Amount10 mL Stripping buffer for Duration00:05:00 using a tubular vessel. (1/2)
5m
Incubate the nitrocellulose membrane twice with Amount10 mL Stripping buffer for Duration00:05:00 using a tubular vessel and a roller mixer. (2/2)
5m
Wash the nitrocellulose membrane three times with Amount10 mL water.

Wash
Wash the nitrocellulose membrane three times with Amount10 mL PBS-T.

Wash
Incubate with Biotin anti-C-tag (Thermo), 1/4000 diluted with 1% skimmed milk powder, in PBS-T, for Duration02:00:00 at TemperatureRoom temperature using a tubular vessel and a roller mixer.

2h
Incubation
Wash the nitrocellulose membrane three times with Amount10 mL PBS-T.

Wash
Incubate with Streptavidin-HRP (Pierce), 1/10000 diluted with 1% BSA in PBS, for Duration01:00:00 at TemperatureRoom temperature using a tubular vessel and a roller mixer.

1h
Incubation
Wash the nitrocellulose membrane three times with Amount10 mL PBS-T.

Wash
Detect marker bands and luminescence signal as in steps 33-36.

Fig. 2 Side-by-side comparison of marker bands (epi-illumination), anti-Clusterin (Clu) and antiC-tag (C-tagged VLDLR-ed) immunoblots. Protein from two gels (top and bottom) was transferred onto one nitrocellulose membrane. The respective concentrations (µM) of VLDLR-ed and Clusterin are indicated above the lanes. The additional lanes show experiments at a different Clu:VLDLR ratio (1:10).