Jan 22, 2024
Ubiquitin immunoprecipitation using an anti-ubiquitin nanobody
- Cole S Sitron1,
- Victoria A Trinkaus1,
- F Ulrich Hartl1
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Protocol Citation: Cole S Sitron, Victoria A Trinkaus, F Ulrich Hartl 2024. Ubiquitin immunoprecipitation using an anti-ubiquitin nanobody. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp36k1vzp/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: January 16, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 93785
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol describes a method to detect ubiquitination on a protein of interest. This technique relies on immunoprecipitation (IP) of ubiquitinated proteins from a cell lysate through the use of an anti-ubiquitin nanobody coupled to agarose beads. The eluate can be run on SDS-PAGE to determine whether the protein of interest was recovered in the IP and, therefore, ubiquitinated.
Attachments
Materials
Materials
Cell culture
2-4 50-80% confluent wells of cells growing in a 6-well plate.
Equipment
Equipment
Bioruptor® Plus sonication device
NAME
Bioruptor®
BRAND
B01020001
SKU
LINK
- CLARIOstar Plus Plate Reader (BMG Labtech) (or equivalent)
- Microcentrifuge
Specialized Reagents
- Ubiquitin SelectorNanoTag BiotechnologiesCatalog #N2510
- MiniSpin Columns (50 units)NanoTag BiotechnologiesCatalog #A1001-L
- TrypLE™ Express Enzyme (1X), phenol redThermo FisherCatalog #12605036
- Pierce™ Rapid Gold BCA Protein Assay KitThermo FisherCatalog #A53225
Buffers
- Triton Lysis Buffer:
| A | B | |
| Tris pH 8 | 20 mM | |
| NaCl | 150 mM | |
| Triton X-100 | 1% | |
| cOmplete, Mini EDTA-free Protease Inhibitor Cocktail (Roche cat. no. 11873580001) | 1X | |
| Freshly-added 20 mM N-ethylmaleimide (2.5 mg/ml; Sigma Aldrich E3876-25G) | 2.5 mg/ml | |
| 50 uM PR-619 (from 100 mM DMSO stock; Sigma-Aldrich 662141-25MG) | 50 uM | |
| Benzonase (Max Planck Institute of Biochemistry Core Facility) | 7.5 U/ml |
cOmplete™ EDTA-free Protease Inhibitor CocktailRocheCatalog #11873580001
N-EthylmaleimideMerck MilliporeSigma (Sigma-Aldrich)Catalog #E3876
DUB Inhibitor V, PR-619Merck MilliporeSigma (Sigma-Aldrich)Catalog #662141
- Triton Wash Buffer:
| A | B | |
| Tris pH 8 | 20 mM | |
| NaCl | 150 mM | |
| EDTA | 1 mM | |
| Triton X-100 | 1% | |
| SDS | 0.1% | |
| PR-619 | 50 uM |
- 4X NuPAGE™ LDS Sample Buffer (4X)Thermo Fisher ScientificCatalog #NP0007 + 5% beta-mercaptoethanol (2-Mercaptoethanol Merck MilliporeSigma (Sigma-Aldrich)Catalog #M6250 -100ml)
- 10% FBS (Fetal Bovine SerumGibco - Thermo FischerCatalog #10270106 ) in 1X PBS (diluted fromPBS (10X), pH 7.4Thermo Fisher ScientificCatalog #70011051 )
- 1X PBS
Cell collection and lysis
Cell collection and lysis
Remove the medium from the wells and trypsinize with 500 µL TrypLE Express.
Quench the TrypLE Express with 500 µL 10% FBS and move the cells into a centrifuge tube.
Pellet cells at 1500 x g for 00:03:00 at 4 °C .
3m
Wash cells with 1 mL PBS and pellet again.
Resuspend cells in 150 µL Triton Lysis Buffer and incubate On ice for 00:05:00 .
5m
Sonicate in the BioRuptor for 5 cycles of 00:00:30 on and 00:00:30 off at 4 °C .
1m
Clarify the lysate by centrifugation 18000 x g for 00:10:00 4 °C .
10m
During the centrifugation, prepare BSA standards according to the Rapid Gold BCA Protein Assay Kit manual and begin equilibrating 50 µL of Ubiquitin pan-selector resin (pipette with a cut p200 tip) in 500 µL of lysis buffer.
Collect supernatant and place into a new tube On ice .
Prepare a small 1:10 dilution of samples and perform Rapid Gold BCA Protein Assay according to manufacturer’s instructions.
Immunoprecipitation (IP)
Immunoprecipitation (IP)
1h 7m
Dilute samples into Triton Lysis Buffer in two tubes: 1 mg in 500 µL (for IP) and 100 µg in 25 µL (for input).
Pellet the beads at 1000 x g for 00:02:00 at 4 °C .
2m
Pull off the supernatant, add the 1 mg of lysate to the beads, and incubate with rotation at 4 °C for 01:00:00 .
1h
During the incubation denature the input sample with 25 µL 4X NuPAGE LDS Sample Buffer at 95 °C 00:05:00 .
5m
Pellet the beads at 1000 x g for 00:02:00 at 4 °C .
2m
Wash the beads with either 1 mL Triton Wash Buffer.
Pellet the beads and repeat for a total of 3 washes.
Remove the supernatant and add 100 µL of 2X NuPAGE LDS Sample Buffer.
Elute by boiling at 95 °C for 00:05:00 .
5m
Transfer the beads to a MiniSpin column placed in a tube.
Centrifuge 3000 x g for 00:03:00 .
3m
The eluate has now been collected in the tube and is ready for SDS-PAGE analysis.