SUMOylation Assay Kit

Product details

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 20 uL 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-K B 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.

Other Notes
The mechanism for SUMO conjugation is analogous to that of the ubiquitin system, relying upon utilization of E1, E2 and E3 cascade enzymes. SUMO modification of target proteins is involved in nuclear protein targeting, formation of sub-nuclear complexes, regulation of transcriptional activities and control of protein stability.

A short sequence containing the consensus Hydrophobic-K-X-D/E (where Lysine is the modified amino acid, Hydrophobic is a large hydrophobic residue and X is any amino acid residue) is thought to be necessary for the protein SUMOylation to occur.