Signal-Seeker™ SUMOylation 2/3 Detection Kit (10 assay)

Signal Seeker SUMOylation 2/3 Detection Kit (10 assays, immunoprecipitation format)
$0.00

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 Include

  • Investigate 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 Paper

Kit contents

The 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 results

There are many applications for these kits, here we describe an interesting example:

Application 1: Investigate significant SUMOylation 2/3 events

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 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):896-909

2. 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.com

Associated 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)

 

Click on the pdf icon below to download the manual

 

     

      For the most recent publications citing this and other Signal-Seeker™ products, see our Signal-Seeker™ Validation Data Page click here

      Visit 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