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
io_Ubiquitin immunoprecipitation using an anti.docx
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.
