Immunoprecipitation using the Signal-Seeker™ SUMOylation 2/3 Detection Kit

Denatured cell lysates were prepared as previously reported¹ from HeLa cells ("HS43": Heat Shock treated 42°C  for 10 min, and "CT37": untreated) and HeLa siRNA SUMO knockdown ("KDS2"). 1mg of lysate was used for the immunoprecipitation (IP) of SUMOylated 2/3 proteins. Western blots of immunoprecipitated proteins were developed using anti-SUMO-2/3 antibody (Cytoskeleton cat# ASM23) (A) or anti-Ubc9 antibody (B). The level of SUMO-2/3 conjugates in heat shock treated cells is higher than control, and shRNA SUMO-2 knock-down reduced the level of SUMOylated 2/3 modified proteins. Chemical conjugation of SUMO-2/3 antibody (11G2) to the affinity bead matrix prevents heavy and light chain leaching. Unconjugated free SUMO is also captured by the SUMO-2/3 affinity beads.

(B) Unmodified Ubc9 is visible near 18kDa. High molecular-weight band indicates that Ubc9 is SUMOylated by a single SUMO-2/3 protein. Ubc9 has previously been reported to be a target for SUMOylation^1,2. 

1. Barysch S. et al. 2014. Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies. Nat Protoc. 9(4):896-909

2. Becker J. et al. 2013. Detecting endogenous SUMO targets in mammalian cells and tissues. Nature Struc. & Mol. Biol. 20, 525-531.