Catalog number: SBB-PS0043, 50 μg
This product consists of a full-length, mature ubiquitin polypeptide (amino acids 1-76) recombinantly expressed in E.coli, conjugated on its c-terminus to AMC. Hydrolysis of the conjugate results in fluorescence observable by excitation at 345nm and emission at 445nm. Typical working range is 50-500nM.
Ubiquitin is a 76 amino acid post-translational modifier expressed throughout all tissues in eukaryotic organisms. The many roles of ubiquitin modification include proteasomal degradation, signal transduction, inflammatory response, and DNA damage repair. Ubiquitin modification occurs through a pyramidal cascade of an E1 activating enzyme, E2 conjugating enzymes ,and an E3 ubiquitin ligases. This enzymatic cascade results in modification of a ε-amine of a lysine residue on a substrate protein. Substrates may either be mono or poly-ubiquitinated by M1, K6, 11, 27, 29, 33, 48 or 63 linkages. Removal of ubiquitin from a substrate protein occurs via deconjugating enzymes, of which there are nearly 100 known enzymes with various specificities.
For Research Use Only, Not For Use In Humans.
|Molecular Weight:||8.9 kDa|
|Purity:||>99% by LCMS|
|Label or Dye:||AMC|
|Substrate Properties:||Protein-Based Substrate|
|Storage Buffer:||50mM MES, pH 6.0|
|Storage||Store at -80˚C. Avoid multiple freeze thaw cycles.|
Figures & Data
Z' & Reaction Progress
Figure 1. Robustness of Ubiquitin-AMC substrates in an HTS format . 500 nM Ub-AMC was incubated with and without 5pM UcHL3 in a 384 well plate (n = 16), and progress curves were normalized to the maximum fluorescence signal to produce, i.e. the “% reaction progress”. The Z’ value, a statistical parameter widely used in the evaluation of screening assays, was calculated at each time-point.
Figure 2. LCMS. Analysis of Ubiquitin-AMC using LCMS intact mass determination indicates purity greater than 99%.
Signal : Background
Figure 3. Signal to Background. The signal to background ratio was determined by 100% hydrolysis of 50nM Ubiquitin-AMC to liberate the quenched conjugate. Assay Buffer: 50mM HEPES pH7.5, 100mM NaCl, 1mM TCEP, 0.1mg/ml BSA.
Certificates of Analysis (COA)
Citations & References
1) Dang, L.C., Melandri, F.D. and Stein, R.L. Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. Biochemistry, 37, 1868-1879 (1998)