SUMOylation Assay Kit (ab139470) provides a means of generating SUMOylated proteins in vitro, by covalent linkage of the carboxy-terminal of SUMO-1, -2 or –3 to specific lysine residues on the target protein via isopeptide bonds, using the SUMOylation enzyme cascade. A control target protein is provided together with all other necessary components. SUMO specific antibodies are provided for detection of SUMOylated proteins via SDS-PAGE and western blotting. This kit provides sufficient material for 20 x 20μL reactions.

Suggested uses for the SUMOylation assay kit include:

1) SUMO-modification of specific proteins in vitro. Allow investigation of the effect SUMOylation has on enzyme function, stabilization, protein:protein interactions and, hence, it’s role in regulation of cellular processes, such as the p53 tumor repressor and NF-_κB pathways.

2) Demonstrate novel proteins are potential targets for SUMOylation under in vitro conditions. Starting point for examining the role SUMOylation of a protein might play in vivo.

3) Generate substrates for deSUMOylating enzymes, such as SENP1 and SENP2.

4) Test proteins for SUMO E3 ligase activity: does it facilitate or enhance SUMOylation of specific target proteins, particularly under conditions/enzyme concentrations that more closely represent those in vivo.

5) Addition of known SUMO E3 ligase to facilitate/enhance target protein SUMOylation, particularly under conditions/enzyme concentrations that more closely represent those in vivo (e.g. RANBP2, shown to be a ligase for SP100 SUMOylation).

6) SUMOylation of proteins in cell lysates or crude fractions/preparations to facilitate investigation of their role/function in complex solutions.

7) Assay SUMOylation of known proteins in specific lysates (confirm with target protein specific antibodies).

8) Use of cell lysate or crude fractions/preparations as source of SUMO E3 ligases to facilitate SUMOylation of purified target proteins in the presence of SUMOylation kit components.