Drummond:Ubiquitin Western

Introduction

Ubiquitin is small; resolving it can be tricky. The following protocol is adapted from John Hanna in Dan Finley's lab.

Protocol

• Prepare logarithmically growing cells.
• Resuspend in 1X LLB at a concentration of 75 uL 1X LLB per 1 OD600 worth of cells.
• Boil 5 min
• Centrifuge briefly to pellet debris.
• Load 15 ul on 4-12% B/T gradient gel (Invitrogen); be sure to use MES buffer as this gives better resolution in the LMW range
• Wet transfer to PVDF for 1 hour at 250 mA (constant)
• Block 40 minutes
• Primary antibody: anti-ubiquitin polyclonal Ab (Biomol: UG9510) 1:2500 in TBS-T for 40 minutes (can re-use this antibody at least 5 times)
• Wash 4x5 min in TBS-T
• Secondary antibody: anti-rabbit IgG Ab 1:10000 in TBT-T for 40 minutes
• Wash 4x5 min in TBS-T
• Wash 1x1 min in water ECL