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Chemical structure for (Ac-ANW)2R110
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1 mg 13455 $195

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Ex/Em (nm)498/520
Storage Freeze (<-15 °C)
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Category Enzyme Detection
Peptidases and Proteases
Related Secondary Reagents
(Ac-ANW)2R110 is a selective fluorogenic substrate for proteasome 20S-beta 5i. The non-fluorescent substrate generates a bright green fluorescent rhodamine 110 product that has Ex/Em = 494/521 nm, and can be easily detected with a FITC filter set. This rhodamine 110 substrate is much more sensitive than the AMC-, AFC- or 4-nitroaniline-based substrates. The most common form of the proteasome is known as the 26S proteasome that contains one 20S core particle structure and two 19S regulatory caps. All 20S particles consist of four stacked heptameric ring structures that are themselves composed of two different types of subunits; alpha subunits are structural in nature, whereas beta subunits are predominantly catalytic. The outer two rings in the stack consist of seven alpha subunits each, which serve as docking domains for the regulatory particles and the alpha subunits N-termini form a gate that blocks unregulated access of substrates to the interior cavity. The inner two rings each consist of seven beta subunits and contain the protease active sites that perform the proteolysis reactions. In mammals, the beta1, beta2, and beta5 subunits are catalytic. Although they share a common mechanism, they have three distinct substrate specificities considered chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide-hydrolyzing. Alternative beta forms denoted beta 1i, beta 2i, and beta 5i can be expressed in hematopoietic cells in response to exposure to pro-inflammatory signals such as cytokines, in particular, interferon gamma.

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of (Ac-ANW)2R110 to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

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Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
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This protocol only provides a guideline, and should be modified according to your specific needs.

1.       Prepare a 5 to 10 mM stock solution in DMSO.

2.       Prepare a 2X proteasome substrate (20 to 50 µM) assay solution as the following:


25 to 50 µL substrate stock solution (10 mM)

100 µL DTT (1M)

400 µL EDTA (100 mM)

10 mL Hepes Buffer (25 mM), pH =7.4


3.       Mix equal volume of the protesome standards or samples with 2X fluorescent proteasome substrate assay solution (from Step 1), and incubate the solutions at room temperature for at least 1 hour.


4.       Monitor the fluorescence use fluorescent microplate readers.

References & Citations

Diabetogenic agent alloxan is a proteasome inhibitor
Authors: Wenjuan Zhou, Lingling Wei, Ting Xiao, Chunyou Lai, Min Peng, Lingli Xu, Xiangwei Luo, Shaoping Deng, Fengxue Zhang
Journal: Biochemical and Biophysical Research Communications (2017): 400--406

Delineation of molecular pathways involved in cardiomyopathies caused by troponin T mutations
Authors: Jennifer E Gilda, Xianyin Lai, Frank A Witzmann, Aldrin V Gomes
Journal: Molecular & Cellular Proteomics (2016): 1962--1981

Diclofenac induces proteasome and mitochondrial dysfunction in murine cardiomyocytes and hearts
Authors: Rajeshwary Ghosh, Sumanta K Goswami, Luis Felipe BB Feitoza, Bruce Hammock, Aldrin V Gomes
Journal: International Journal of Cardiology (2016): 923--935

Advanced-glycation-end-product-induced formation of immunoproteasomes: involvement of RAGE and Jak2/STAT1
Authors: Stefanie Grimm, Christiane Ott, Melanie Hörlacher, Daniela Weber, Annika Höhn, Tilman Grune
Journal: Biochemical Journal (2012): 127--139

Additional Documents

Safety Data Sheet (SDS)

1. Enzyme Probes & Assay Kits

Certificate of Analysis