Description
Key facts
Sample types:Purified protein,
Assay type:Enzyme activity
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.
Properties and Storage Information:
Shipped at conditions-Dry Ice, Appropriate short-term storage conditions--80°C, Appropriate long-term storage conditions--80°C, Storage information--80°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Protein sumoylation also known as post-translational modification involves the attachment of small ubiquitin-like modifier (SUMO) proteins to target proteins. SUMO proteins are about 11 kDa in mass. This process alters the target proteins' functions localization and interactions. SUMO proteins express widely in eukaryotic cells influencing cellular processes and regulatory pathways. Sumoylation machinery includes enzymes like E1 E2 and E3 which add SUMO to lysine residues on substrates.
Biological function summary
The sumoylation process acts as a regulatory mechanism within the cell similar to phosphorylation. It participates in various cellular activities such as nuclear transport transcriptional regulation and DNA repair. SUMO proteins collaborate with other components to form protein complexes influencing cellular homeostasis and stress responses. Cells rely on sumoylation to maintain dynamic balance integrating environmental signals to modulate gene expression and protein stability.
Pathways
Protein sumoylation plays substantial roles in critical pathways like the nuclear import and DNA damage response pathways. It regulates proteins such as RanGAP1 and PCNA which are essential in these pathways. The modification allows proteins to interact or avoid interacting with certain partners impacting cellular signaling and repairing activity. Sumoylation also works through crosstalk with other modifications allowing cells to fine-tune responses to various signals.
Sumoylation dysfunction associates with conditions such as cancer and neurodegenerative diseases. Abnormal sumoylation affects proteins like p53 and tau altering their usual roles and contributing to pathological states. For instance in cancer improper modification of p53 can disrupt its function as a tumor suppressor. In neurodegenerative diseases altered tau sumoylation affects its aggregation state linking to disease progression. Understanding sumoylation in these contexts opens potential therapeutic approaches for these conditions.
