Details The Signal-Seeker™ line of produts have been developed to allow simple analysis of key regulatory protein modifications by specialists and non-specialists alike. The comprehensive Signal-Seeker™ kits provide an affinity bead system to isolate and enrich modified proteins from any given cell or tissue lysate. The enriched protein population is then analyzed by standard western blot procedures using a primary antibody to the target protein. Product Uses IncludeInvestigate transient regulatory mechanisms Measure signalling events of multiple pathway member proteins  Discover new modifications of your protein of interest Gain insight into regulatory mechanisms Measure endogenous or transiently expressed protein signalling events Validation Data: SUMOylation 2/3 Detection Kit White PaperKit contentsThe SUMOylation 2/3 kit contains the following components:Lysis and protein quantitation stepIP and pre-clear stepWash stepElution stepWestern step BlastR™ Lysis Buffer  BlastR™ Dilution Buffer BlastR™ Filters Protease Inhibitor Cocktail De-SUMOylation inhibitor  Cocktail Precision Red™ Protein  Assay Reagent SUMOylation 2/3  Affinity Beads IP Control  Beads     BlastR™ Wash  Buffer      Spin Columns Bead Elution  Buffer     Chemiluminescent  Reagent A Chemiluminescent  Reagent B Anti-SUMO2/3-HRP  antibody    Example resultsThere are many applications for these kits, here we describe an interesting example:Application 1: Investigate significant SUMOylation 2/3 eventsImmunoprecipitation using the Signal-Seeker™ SUMOylation 2/3 Detection KitDenatured 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 SUMOylation1,2. 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):896-9092. Becker J. et al. 2013. Detecting endogenous SUMO targets in mammalian cells and tissues. Nature Struc. & Mol. Biol. 20, 525-531.Other experiments that could be attempted in this area of research include:• Pharmacological investigation of SUMOylating  and de-SUMOylating enzymes involved in regulation of target proteins.• Investigate SUMOylation under a variety of different growth factors or drug treatments.• Examine the interaction of SUMOylated target proteins with its downstream effectors.• Examine crosstalk between SUMOylation 2/3 and other PTMs for target proteins. For more information contact:  signalseeker@cytoskeleton.comAssociated Products:Signal-Seeker™ Phosphotyrosine Detection Kit (Cat. # BK160)Signal-Seeker™ Ubiquitination Detection Kit (Cat. # BK161)Signal-Seeker™: BlastR™ Rapid Lysate Prep Kit (Cat. # BLR01)Signal-Seeker™ SUMOylation 2/3 Affinity Beads (Cat.# ASM24-beads)Signal-Seeker™: PTMtrue™ SUMOylation 2/3 Antibody (Cat.# ASM23)About Click on the pdf icon below to download the manual   CitationsFor the most recent publications citing this and other Signal-Seeker™ products, see our Signal-Seeker™ Validation Data Page click hereFaqsVisit our Signal-Seeker™ Tech Tips and FAQs page for technical tips and frequently asked questions regarding this and other Signal-Seeker™ products click here If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com