K-930

Material Data Sheet

Fraction II Conjugation Kit

Price: $295.00

Catalog #: K-930

Fraction II Conjugation Kit

Price:

$295

Quantity:

1 kit

Data Sheet:

K930_FIIHeLa conj.pdf

Stock:

1. Fraction II HeLa, 250 μL
    [HeLa] = X mg/ml in 50 mM HEPES, pH 7.6, 1 mM DTT2. Ubiquitin, 10 X stock, 75 μL
    [Ub] = X mg/ml in 50 mM HEPES, pH 7.63. Energy Regeneration Solution (ERS), 10X, 50 μL4. Ubiquitin Aldehyde, 20 μg, solid5. MG-132, Inhibitor Control, 20 μL
    [MG-132] =X μM in 100% DMSO

Background:

The Ubiquitin Proteasome Pathway (UPP) is the cell’s principle mechanism for protein catabolism. The UPP has been shown to have significant involvement in the regulation of critical cellular processes such as transcription, oncogenesis, cell cycle progression, development, growth, selective elimination of abnormal proteins, and antigen processing. The covalent bond between ubiquitin and substrate proteins requires the activation of the C-terminal carboxyl group of Ub to be activated. In this reaction, ubiquitin is linked to the Ubiquitin Activating Enzyme (E1) through a thioester bond between its C-terminal Gly and the active site cysteine residue in the enzyme (E1-S-Ub). E1 hydrolyzes ATP to AMP and PPi with the intermediate formation of an E1-bound ubiquitin adenylate. The activated Ub is then transferred from E1 to any of a family of Ubiquitin Carrier Protein Enzymes (E2s) in a similar thioester linkage (E2-S-Ub). Ubiquitin can then be transferred directly to the ε-Lys of a substrate from E2 enzymes. Other conjugation reactions require the involvement of a third enzyme, Ubiquitin Ligase (E3). E3 binds to substrate proteins and also to an E2 to form an E2-E3- substrate complex. It is the recognition and formation of said complex that has the highest level of substrate specificity for the conjugation cascade.

This kit contains reagents to allow for the controlled conjugation of exogenously added or endogenously contained substrate proteins through the ubiquitin proteasome pathway (UPP). Fraction II HeLa is the protein fraction of a cell extract that binds to anion exchange resin. It is essentially free of ubiquitin and ATP. FII contains E1, most E2s, some E3s, UCHs and the proteasome (20S and 26S).

NOTE: Kit contains reagents sufficient for 10 x 50 μl reactions.

Storage:

Enzyme/Fraction/Protein Solutions should be stored at -80°C. Avoid multiple freeze/thaw cycles.

Literature

References:

Ciechanover A. (1998) EMBO. J. 17:7151-7160
Hershko A. and Ciechanover A. (1998) Ann. Rev. Biochem. 67:425-479
Glickman M.H and Ciechanover A. (2001 Physiol. Rev. 82:373-428
Schwartz A.L. and Ciechanover A. (1999) Ann. Rev. Med. 50:57-74