Product Spotlight: Ubiquitinated Protein Enrichment Kit  |
Cat. No. 662200
A vast majority of short-lived proteins are degraded by the ubiquitin proteasome pathway. A protein marked for degradation is covalently attached to multiple molecules of ubiquitin, a highly conserved 76-amino acid (8.6 kDA) protein, by a multi-enzymatic system consisting of Ubiquitin-activating (E1), Ubiquitin-conjugating (E2), and the Ubiquitin-ligating (E3) enzymes. The E1 activates a Ubiquitin monomer at its C-terminal cysteine residue to a high-energy thiolester bond which is then transferred to a reactive cysteine residue of the E2 enzyme. Ubiquitinated protein is then escorted to the 26S proteasome where it undergoes final degradation and the ubiquitin is released and recycled. A family of proteins including Rad23, contain two ubiquitin-associated domains that bind ubiquitinated cellular proteins and translocate them to the proteasome. Ubiquitinated proteins can be enriched using affinity beads comprised of a GST-fusion protein containing this ubiquitin-associated sequence conjugated to glutathione -agarose.
This kit is useful for the enrichment of polyubiquitinated proteins from cell and tissue lysates of a broad range of species including canine, human, mouse, and yeast (see figure below).
The ubiquitated proteins can be identified by loading the beads directly onto SDS-PAGE and then immunoblotting with the antibody of choice or Anti-Ubiquitin ( Cat. No. 662099
). Alternatively, it is possible that the beads can first be treated with Isopeptidase T ( Cat. No. 419700
) to release the proteins from the ubiquitin chains.
Each kit contains polyubiquitin affinity beads, control glutathione-agarose beads, control lysate, and an instructional protocol. (Quantities sufficient for 25 affinity purification assays)
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