Jul 01, 2025
Ubiquitin enrichment assay
- Bishal Basak1,2,
- Erika Holzbaur1,2
- 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
- University of Pennsylvania
Protocol Citation: Bishal Basak, Erika Holzbaur 2025. Ubiquitin enrichment assay. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly4z7rlx9/v1
Manuscript citation:
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 30, 2025
Last Modified: July 01, 2025
Protocol Integer ID: 221323
Keywords: ubiquitin enrichment, neuronal lysate, assay
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000350
Abstract
Ubiquitin enrichment assay from neuronal lysates
Protocol materials
Signal-Seeker Ubiquitination Detection KitCytoskeleton Inc.Catalog #BK161
Troubleshooting
Plate neurons
Plate 3- 5 million neurons in a 10 cm dish at days in vitro (DIV) 0
Assay steps
When neurons are mature (DIV 6 or more), treat neurons depending on experimental needs
Post treatment, aspirate out media, wash neurons once with 1X fresh PBS
Lyse neurons in lysis buffer containing 50 mM HEPES (pH=7.4), 1 mM EDTA, 1 mM MgCl2, 25 mM NaCl, 0.5% Triton X-100, 20μg/mL Leupeptin, 2 mM DTT, 20 μg/mL TAME, 2 μg/mL Pepstatin A, 1 mM PMSF. Add Deubiquitination inhibitors provided with Ubiquitin Detection Kit (Cytoskeleton, BK161) to the lysis buffer at 1X concentration.
Signal-Seeker Ubiquitination Detection KitCytoskeleton Inc.Catalog #BK161
Post lysis of the neurons, spin lysates at 17000x g for 10 mins, and discard the pellet.
Store 5% of the supernatant as input to be used for western blotting later.
Wash Ubiquitination affinity beads and Ubiquitination IP control beads (provided in the kit) with 1XPBST twice and 1XPBS once, and then incubate with equal volumes of the lysate for 4 hrs at 40C.
Note
The assay was performed using Signal Seeker Ubiquitination Detection Kit, as mentioned in the manufacturer's protocol with some modifications. Signal-Seeker Ubiquitination Detection KitCytoskeleton Inc.Catalog #BK161
Post incubation, centrifuge beads at 5000x g at 40C for 1 min.
Wash beads thrice with BlastR-2TM wash buffer for 5 minutes each in a rotating platform at 40C and centrifuge at 5000Xg.
After final wash and centrifugation, remove buffer supernatant completely.
Incubate beads with Bead Elution Buffer for 5 mins at RT and then centrifuge in a spin column at 10000x g for 1 min to collect the immunoprecipitates.
Add 2-mercaptoethanol to each sample and boil samples at 950C for 10 minutes to be used for western blotting.