Ubiquitin

Ubiquitin is a 76-amino acid polypeptide expressed in all eukaryotic cells and it is highly conserved from yeast to humans. The modification of proteins by chains of ubiquitin has long been known to mediate targeting of cytosolic and nuclear proteins for degradation by proteasomes. Ubiquitination is an essential cellular process affected by a multi-enzyme cascade involving classes of enzymes known as ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s or Ubcs) and ubiquitin-protein ligases (E3s). E1 activates ubiquitin in an ATP-dependent manner with the formation of a thiol-ester linkage between the carboxy terminus of ubiquitin and E1. Sequential, transient thiol-ester bonds are then generated between the carboxyl terminus of ubiquitin and specific cysteines of E2 and E3 enzymes. These bonds culminate in the formation of an isopeptide linkage between the activated carboxy terminus of ubiquitin and the epsilon-amino group of a lysine on a target protein or within another ubiquitin chain (usually K48 of ubiquitin) resulting in the generation of chains of ubiquitin (polyubiquitin or multiubiquitin) which may be from 4–20 ubiquitin moieties.

Applications:
Suitable for use in Western Blot, Immunohistochemistry and Immunocytochemistry. Other applications not tested.

Recommended Dilutions:
Western Blot (ECL): 1:5000
Immunohistochemistry: Paraffin sections
Optimal dilutions to be determined by the researcher.

Positive Controls:
Rat Brain Tissue Extract and Heat-shocked HeLa Cell Lysate

Storage and Stability:
May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 12 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.