NEDD8-AMC

Catalog number: SBB-PS0044, 50 μg

This product consists of a full-length, mature NEDD8 polypeptide 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
0044
$199.00

Description

NEDD8 (Neural Precursor Cell Expressed, Developmentally Down-Regulated 8) is a ubiquitin like protein that plays an important role in regulating development and the cell cycle.  NEDD8 is conjugated to a target protein via a signaling cascade similar to ubiquitin: a NEDD8 specific E1 activating enzyme (APPBP1/UBA3) adenylates the c-terimnus of NEDD8 which is than subsequently passed to an E2 conjugating enzyme (UBE2M or UBE2F) that facilitates covalent attachment of NEDD8 to a cullin subunit of an SCF E3 ubiquitin ligase. NEDD8-Rh 110 can be used as a substrate for enzymes exhibiting deNEDDylating activity, e.g UCH-L3, UCH-L1, COP9 Signalosome, and NEDP1.  This product consists of a full-length, mature NEDD8 polypeptide (amino acids 1-76) recombinantly expressed in E.coli, conjugated on its c-terminus to a quenched AMC110 dye.  Once hydrolyzed the free AMC provides excellent utility for real time assessment of enzyme activity at excitation (345 nm) and emission (445 nm). Typical working range is 50-500nM.

For Research Use Only, Not For Use In Humans.

Specifications

Product Overview
Quantity: 50 μg
Molecular Weight: 8.7 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 NEDD8-AMC substrates in an HTS format . Fluorescent substrate NEDD8-AMC was incubated with and without 30 pM NEDP1 in a 384 well plate (n = 16), and progress curves were normalized to the maximum fluorescence signal to produce “% reaction progress”.  The Z’ value, a statistical parameter widely used in the evaluation of screening assays, was calculated at each timepoint.  

Mass Spectrometry

Figure 2. LCMS. Analysis of Ubiquitin-AMC using LCMS intact mass determination (8,717 kDa) indicates purity greater than 99%.

Signal : Background

Figure 3. Signal to Background. The signal to background ratio was determined by 100% hydrolysis of 50nM NEDD8-AMC to liberate the quenched conjugate. Assay Buffer: 50mM HEPES pH7.5, 1mM TCEP, 0.1mg/ml BSA.

Certificates of Analysis (COA)

Citations & References

1) Walden H, Podgorski MS, Huang DT, Miller DW, Howard RJ, Minor DL, Holton JM, Schulman BA (2003). “The structure of the APPBP1-UBA3-NEDD8-ATP complex reveals the basis for selective ubiquitin-like protein activation by an E1”. Mol. Cell. 12 (6): 1427–37. PMID 14690597 2) Brown JS, Lukashchuk N, Sczaniecka-Clift M, Britton S, le Sage C, Calsou P, Beli P, Galanty Y, Jackson SP (2015). “Neddylation promotes ubiquitylation and release of Ku from DNA-damage sites”. Cell Rep. 11 (5): 704–14. doi:10.1016/j.celrep.2015.03.058. PMID 25921528.