Jan 13, 2025
Pulldown of protein aggregates with a biotinylated peptide
- Cole Sitron1,
- Patricia Yuste Checa1,
- Victoria A Trinkaus1,
- F Ulrich Hartl1
- 1Max Planck Institute of Biochemistry
Protocol Citation: Cole Sitron, Patricia Yuste Checa, Victoria A Trinkaus, F Ulrich Hartl 2025. Pulldown of protein aggregates with a biotinylated peptide. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5wrwg47/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: June 10, 2024
Last Modified: January 13, 2025
Protocol Integer ID: 102730
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000282
Aligning Science Across Parkinson’s
Grant ID: ASAP- 024268
Abstract
The purpose of this protocol is to test whether monomeric or aggregated versions of a protein can bind to a peptide. This is tested by performing a pulldown using an affinity reagent that consists of a biotinylated peptide conjugated to streptavidin beads. This protocol describes how to perform this pulldown with α-syn and Aβ42 monomers and aggregates as prey, but the same pulldown protocol could be extended to any prey of interest. The endpoint of this protocol is the production of input, flow-through, and eluate fractions from the pulldown, which can be analyzed by SDS-PAGE and/or immunoblot.
Materials
- Pierce Magnetic Streptavidin Beads
- Biotinylated peptide of interest
- Peptide Solubilization Buffer: 45% ethanol, 10% acetic acid, 1 mM DTT in ddH2O.
- 330 µM α-syn A53T monomers, prepared as in dx.doi.org/10.17504/protocols.io.btynnpve.
- 330 µM α-syn A53T aggregates (PFFs), prepared as in dx.doi.org/10.17504/protocols.io.btynnpve.
- Lyophilized Aβ42 protein, prepared exactly as in described in the “without urea” protocol in Linse 2020 (Section 3.6)
- Alternatively, this protein can be bought, e.g. Thermo Fisher Scientific cat. no. 03-111
- Blocking Buffer:
PBS, pH 7.2Thermo Fisher ScientificCatalog #20012068 , 1% Triton™ X-100 Surfact-Amps™ Detergent SolutionThermo FisherCatalog #85111 , additional 113 mM NaCl (final concentration 250 mM), 3%BSACell Signaling TechnologyCatalog #9998S
- IP Buffer: PBS pH 7.2, 1% Triton X-100, additional 113 mM NaCl (final concentration 250 mM), 0.1% SDS, 1 mM DTT
- 500 mM NaCl in ddH2O.
- GE Buffer: 6M GuHCl, 20 mM sodium phosphate, pH 8.5
- Aβ42 Aggregation Buffer: 20mM sodium phosphate pH 8.5, 0.2 mM EDTA
- NUPAGE LDS sample buffer (4x)Thermo Fisher ScientificCatalog #NP0007 + 5% 2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
- HU Buffer: 8 M urea, 5% SDS, 200 mM Tris-HCl pH 6.8, 1 mM EDTA, 0.01% bromophenol blue, 5% β-mercaptoethanol
- Formic Acid
- Superdex 75 Column (GE HealthCare cat. no. GE17-5174-01)
- FPLC Setup
- Microcentrifuge
- Magnetic tube rack
- Tube rotator
- Bioruptor Plus sonication systemDiagenodeCatalog #B01020001 (or equivalent)
- Nanodrop
- Thermomixer
- SpeedVac
- Protein LoBind 1.5mL microcentrifuge tubesEppendorfCatalog #0030108116
- 96-well Half Area Black/Clear Flat Bottom Polystyrene (Corning cat. no. 3881)
Solubilization and Aggregation of Aβ42
Solubilization and Aggregation of Aβ42
1d
1d
Equilibrate a Superdex 75 column with Aβ42 Aggregation Buffer.
Resuspend 500 µg of lyophilized Aβ42 in 700 µL GE Buffer.
Use an FPLC setup to run the solubilized Aβ42 through the Superdex 75 column, collecting the fractions in low bind tubes.
Check the fractions by 205 nanomolar (nM) absorbance on a Nanodrop and pool the Aβ42-containing fractions.
Dilute the Aβ42 to 10 micromolar (µM) in Aβ42 Aggregation Buffer.
Aliquot and snap freeze half of the sample to use later as Aβ42 monomers.
Place 80 µL in each well of a 96-well plate and incubate for 24:00:00 at 37 °C .
1d
Aliquot the aggregated material, flash freeze, and store at -80 °C .
Conjugation of the biotinylated peptide to Streptavidin resin (bait)
Conjugation of the biotinylated peptide to Streptavidin resin (bait)
2h
2h
Dissolve in Peptide Solubilization Buffer to an appropriate concentration.
In our case, we resuspend our peptide to 2 µL which was 390.6 micromolar (µM) .
Add 25 µL of Pierce Magnetic Streptavidin Beads to a low bind tube and place on the magnetic rack.
Be sure to pipette with a cut pipette tip to avoid damaging the resin.
Prepare as twice as many tubes as prey proteins that will be pulled down. One tube will be for the normal pulldown and the other will be a no peptide control to assess background binding.
Wash the beads once with 1 mL IP Buffer.
Resuspend the beads in 400 µL IP Buffer.
Add 3.2 µL of the solubilized biotinylated peptide to the half of the tubes with beads.
To the other half add the same volume of Peptide Solubilization Buffer.
And rotate the tube for 01:00:00 at Room temperature to allow the biotinylated peptide to complex with the streptavidin.
1h
Wash the beads twice with 500 µL of 500 millimolar (mM) NaCl, then once with 1 mL IP buffer.
Incubate the beads with rotation in 500 µL Blocking Buffer for 01:00:00 .
1h
Wash the beads once with IP Buffer.
Preparation of protein aggregates and monomeric controls (prey) for pulldown
Preparation of protein aggregates and monomeric controls (prey) for pulldown
31m 10s
31m 10s
Thaw the Aβ42 aggregates, α-syn monomers, and α-syn PFFs On ice .
Centrifuge the Aβ42 and α-syn monomers at 20000 x g, 4°C, 00:30:00 to remove any aggregated material.
30m
Remove the supernatant and place in a new tube.
Sonicate the α-syn PFFs in the Bioruptor for 5 cycles of 00:00:30 on and 00:00:30 off.
1m
Sonicate the Aβ42 aggregates for 5 cycles of 00:00:05 on and 00:00:05 off.
10s
Dilute each sample to 2.5 micromolar (µM) in IP Buffer.
Pulldown
Pulldown
1h 0m 30s
1h 0m 30s
Add 400 µL of 2.5 micromolar (µM) Aβ42 monomers, Aβ42 aggregates, α-syn monomers, and α-syn to appropriate tubes.
Each sample should go into one tube with peptide-conjugated beads and into one peptide-free control tube.
Reserve a small amount of the 2.5 micromolar (µM) sample to run as an input fraction.
Rotate the tubes for 01:00:00 at Room temperature , except for the Aβ42 monomers, which need to be incubated at 4 °C to prevent aggregation.
1h
Gently centrifuge the tubes (∼50 x g, 00:00:30 ) to remove the resuspended beads from the cap area of the tube.
30s
Place the tube on the magnetic rack and set aside the supernatant to run as a flow-through fraction.
Using the magnetic rack, wash the beads three times with 500 µL IP Buffer.
On the last wash, transfer to a new tube.
Normal Elution (appropriate for α-syn)
Normal Elution (appropriate for α-syn)
10m
10m
Replace the IP Buffer with 50 µL 4X NuPAGE LDS Sample Buffer + 5% β-mercaptoethanol.
Gently flick the tube to resuspend the resin.
Boil the tube for 00:10:00 at 95 °C .
10m
Use the magnetic rack to remove the supernatant, which will be the eluate fraction.
The input and flow-through fractions can now be denatured by boiling with the NuPAGE LDS Sample Buffer + β-mercaptoethanol (diluted 1:4).
Harsh Elution (appropriate for Aβ42)
Harsh Elution (appropriate for Aβ42)
1h 50m
1h 50m
Replace the IP Buffer with 100 µL formic acid.
Add 90 µL formic acid to 10 µL of the input and flow-through fractions.
Incubate at 19 °C for 00:40:00 with shaking at 500 rpm in a Thermomixer.
40m
Use the magnetic rack to remove the supernatant from the beads and place the supernatant in a new tube.
Evaporate the all fractions containing formic acid in a SpeedVac for 01:00:00 at 45 °C .
1h
Add 50 µL HU Buffer + 5% β-mercaptoethanol to the tubes and incubate for 00:10:00 at 60 °C shaking at 500 rpm in a thermomixer.
10m
Protocol references
1. Linse, S. (2020). Expression and Purification of Intrinsically Disordered Aβ42 Peptide and Setup of Reproducible Aggregation Kinetics Experiment. In: Kragelund, B.B., Skriver, K. (eds) Intrinsically Disordered Proteins. Methods in Molecular Biology, vol 2141. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0524-0_38