Material Data Sheet
S-100 Fraction Conjugation Kit
S-100 Fraction Conjugation Kit
This kit contains reagents to allow for the controlled conjugation of exogenously added or endogenously contained substrate proteins through the ubiquitin proteasome pathway (UPP). S-100 fraction contains the full complement of all UPP enzymes in the cytosol and is ideal for the demonstration of a protein being targeted by the UPP for degradation..
NOTE: Please read this instruction material completely prior to performing the assay.
Stock:1. S-100 HeLa, X μL [S-100] = X mg/ml. Buffer: 50 mM HEPES, pH 7.6, 1 mM DTT2.Ubiquitin, 10 X stock, X μL [Ub] = X mg/ml. Buffer: 50 mM HEPES, pH 7.63. 10X Energy Regeneration Solution (ERS), X μL4. Ubiquitin Aldehyde, Xμg [Ub-H] = X mg/ml in 50mM HCl5.MG-132 Inhibitor Control, X 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.<br>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 glycine 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 E2s (ubiquitin carrier protein enzymes) 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 ligases (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.
Recommended Assay Protocol:The S100 HeLa fraction contains all the necessary components to ubiquitinate and then degrade UPP substrates. These enzymes/proteins include ubiquitin, E1, E2s, E3s (ligases), isopeptidases (UCHs) and proteasome (26S). Conjugation of the proteins of interest, if already contained in the HeLa fraction may be tracked via immuno-detection or if added exogenously, by immuno-detection or radio-label. When tracking the increase of ubiquitinated products in a SDS-PAGE band is your approach, care must be taken to add substrate to an amount that gives a clearly defined signal. The ultimate optimal amount of substrate to be added must be determined experimentally. If the substrate protein is already contained in the S-100, HeLa and is being assayed by immuno-detection, this is not an issue. The following assay protocol is for the set-up and execution of the experimental protein conjugation. Final concentrations of substrate and time courses must be determined experimentally for each individual protein. Detection methods must also be optimized based on activity, substrate quantity and time. The removals of ERS, ubiquitin or inhibitors are viable negative controls for demonstration of a UPP mediated event. Quantities of supplied reagents or reaction volumes may be changed to suit individual needs.
Enzyme/Fraction/Protein Solutions should be stored at -80°C. Avoid multiple freeze/thaw cycles.
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