SUMOylation Assay: An In Vitro Technique to Detect the SUMOylation Status of Substrate Proteins by Immunoblotting
내레이션 대본
SUMOylation occurs due to the attachment of small ubiquitin-like modifiers, SUMOs, to substrate proteins.
SUMOylation occurs when a mature SUMO peptide is activated by an enzyme – E1. Upon activation, the SUMO is transferred to the active site of the conjugating enzyme – E2 – attaching the SUMO to the lysine residue of the substrate protein. Another ligase – E3 – enhances this interaction's efficiency.
To perform an in vitro SUMOylation assay, begin with a tube containing a mixture of the substrate protein – p53, enzymes, and SUMO peptides, and incubate to induce SUMOylation. Supplement the tube with sodium dodecyl sulfate – SDS-containing buffer, terminating the reaction. Heat the sample to denature the proteins.
Load the protein mix into the well of an SDS-PAGE gel and perform electrophoresis. Higher molecular weight SUMOylated proteins move slower than their non-SUMOylated variants, generating two distinct bands.
Transfer the gel onto a blotting membrane present on a wet filter paper in an electroblotting apparatus. Cover with a wet filter paper and electro-transfer the proteins from the gel onto the membrane.
Treat the membrane with anti-p53 antibodies, which bind to p53. Overlay with enzyme-conjugated secondary antibodies that bind to antibody-p53 complexes. Add a chemiluminescent substrate to generate luminescent product and image the membrane.
A higher molecular weight band corresponds to SUMOylated proteins, while the lower band corresponds to non-SUMOylated protein variants.
