Ubiquitin immunoprecipitation using an anti-ubiquitin nanobody
F Ulrich Hartl, Cole S Sitron, Victoria A Trinkaus
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
Steps
Cell collection and lysis
Remove the medium from the wells and trypsinize with 500µL TrypLE Express.
Quench the TrypLE Express with 500µL 10% FBS and move the cells into a centrifuge tube.
Pellet cells at 1500x g,0h 0m 0s for 0h 3m 0s at 4°C.
Wash cells with 1mL PBS and pellet again.
Resuspend cells in 150µL Triton Lysis Buffer and incubate On ice for 0h 5m 0s.
Sonicate in the BioRuptor for 5 cycles of 0h 0m 30s on and 0h 0m 30s off at 4°C.
Clarify the lysate by centrifugation 18000x g,0h 0m 0s for 0h 10m 0s 4°C.
During the centrifugation, prepare BSA standards according to the Rapid Gold BCA Protein Assay Kit manual and begin equilibrating 50µL of Ubiquitin pan-selector resin (pipette with a cut p200 tip) in 500µL 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)
Dilute samples into Triton Lysis Buffer in two tubes: 1mg in 500µL (for IP) and 100µg in 25µL (for input).
Pellet the beads at 1000x g,0h 0m 0s for 0h 2m 0s at 4°C.
Pull off the supernatant, add the 1mg of lysate to the beads, and incubate with rotation at 4°C for 1h 0m 0s.
During the incubation denature the input sample with 25µL 4X NuPAGE LDS Sample Buffer at 95°C 0h 5m 0s.
Pellet the beads at 1000x g,0h 0m 0s for 0h 2m 0s at 4°C.
Wash the beads with either 1mL Triton Wash Buffer.
Pellet the beads and repeat for a total of 3 washes.
Remove the supernatant and add 100µL of 2X NuPAGE LDS Sample Buffer.
Elute by boiling at 95°C for 0h 5m 0s.
Transfer the beads to a MiniSpin column placed in a tube.
Centrifuge 3000x g,0h 0m 0s for 0h 3m 0s.
The eluate has now been collected in the tube and is ready for SDS-PAGE analysis.
