Ubiquitin-AMC

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.

In stock
SKU
0043
$149.00

Description

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.

Specifications

Product Overview
Quantity: 50 μg
Molecular Weight: 8.9 kDa
Purity: >99% by LCMS
Readout: Endpoint/Kinetic
Label or Dye: AMC
Detection Method: Fluorescence
Substrate Properties: Protein-Based Substrate
Excitation/Emission (nm): 345/445
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. 

Mass Spectrometry

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)