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AAT Bioquest

(Ac-WLA)2R110

The non-fluorescent R110 substrates generate the bright green fluorescent rhodamine 110 product that has Ex/Em = 494/521 nm, and can be easily detected with a FITC filter set. In general, R110 substrates are much more sensitive than the AMC-, AFC- or 4-nitroaniline-based substrates. This R110 substrate is used for monitoring the protease activities of the proteasome. 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. AAT Bioquest offers a group of R110 substrates for monitoring the protease activities of the proteasome at different subsites, i.e., (i) sub-sites: beta1c, Z-LLE-R110; beta2c, Ac-KQL-R110; beta5c, Ac-WLA-R110; beta1i, Ac-PAL-R110; beta2i, Ac-KQL-R110; beta5c, Ac-WLA-R110 and Suc-LLVY-R110; and beta5i, Ac-ANW-R110. The protease activity is measured by monitoring the R110 liberation over time using excitation and emission wavelengths of 490 nm and 520 nm respectively.

Example protocol

AT A GLANCE

Important notes
Following protocol only provides a guideline, and should be modified according to your specific needs.

Product Number

Indicators

Excitation

Emission

13451

(Suc-LLVY)2R110

498 nm

520 nm

13453

Suc-LLVY-AMC

351 nm

430 nm

13455

(Ac-ANW)2R110

498 nm

520 nm

13465

(Ac-KQL)2R110

498 nm

520 nm

13466

(Z-LLE)2R110

498 nm

520 nm

13467

(Ac-PAL)2R110

498 nm

520 nm

13468

(Ac-WLA)2R110

498 nm

520 nm

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. (Ac-WLA)2R110 stock solution (10 mM):
Add 85 µL of DMSO into the vial of 1 mg (Ac-WLA)2R110 to make stock solution (10 mM).

PREPARATION OF WORKING SOLUTION

Proteosome assay solution (2X) (20-40 µM):
Prepare a 20-40 µM of 2X proteasome assay solution in 25 mM HEPES buffer pH 7.5, containing 5mM EDTA, 2mM DTT, and  2% Triton X-100.

SAMPLE EXPERIMENTAL PROTOCOL

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

  2. Monitor the fluorescence using fluorescent microplate readers at 490/525 nm.

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)
(Ac-ANW)2R11050052280000
(Ac-KQL)2R11050052280000
(Ac-PAL)2R11050052280000

Citations

View all 4 citations: Citation Explorer
Diabetogenic agent alloxan is a proteasome inhibitor
Authors: Zhou, Wenjuan and Wei, Lingling and Xiao, Ting and Lai, Chunyou and Peng, Min and Xu, Lingli and Luo, Xiangwei and Deng, Shaoping and Zhang, Fengxue
Journal: Biochemical and Biophysical Research Communications (2017): 400--406
Delineation of molecular pathways involved in cardiomyopathies caused by troponin T mutations
Authors: Gilda, Jennifer E and Lai, Xianyin and Witzmann, Frank A and Gomes, Aldrin V
Journal: Molecular & Cellular Proteomics (2016): 1962--1981
Diclofenac induces proteasome and mitochondrial dysfunction in murine cardiomyocytes and hearts
Authors: Ghosh, Rajeshwary and Goswami, Sumanta K and Feitoza, Luis Felipe BB and Hammock, Bruce and Gomes, Aldrin V
Journal: International Journal of Cardiology (2016): 923--935
Advanced-glycation-end-product-induced formation of immunoproteasomes: involvement of RAGE and Jak2/STAT1
Authors: Grimm, Stefanie and Ott, Christiane and Hörlacher, Melanie and Weber, Daniela and Höhn, Annika and Grune, Tilman
Journal: Biochemical Journal (2012): 127--139

References

View all 36 references: Citation Explorer
Alveolar extracellular 20S proteasome in patients with acute respiratory distress syndrome
Authors: Sixt SU, Adamzik M, Spyrka D, Saul B, Hakenbeck J, Wohlschlaeger J, Costabel U, Kloss A, Giesebrecht J, Dahlmann B, Peters J.
Journal: Am J Respir Crit Care Med (2009): 1098
Quinone reductase acts as a redox switch of the 20S yeast proteasome
Authors: Sollner S, Schober M, Wagner A, Prem A, Lorkova L, Palfey BA, Groll M, Macheroux P.
Journal: EMBO Rep (2009): 65
Comparative expression analysis and characterization of 20S proteasomes in human intestinal tissues: The proteasome pattern as diagnostic tool for IBD patients
Authors: Visekruna A, Joeris T, Schmidt N, Lawrenz M, Ritz JP, Buhr HJ, Steinhoff U.
Journal: Inflamm Bowel Dis (2009): 526
Enzymatic properties of the 20S proteasome in wheat endosperm and its biochemical characteristics after seed imbibition
Authors: Shi C, Rui Q, Xu LL.
Journal: Plant Biol (Stuttg) (2009): 849
Toward a full characterization of the human 20S proteasome subunits and their isoforms by a combination of proteomic approaches
Authors: Uttenweiler-Joseph S, Claverol S, Sylvius L, Bousquet-Dubouch MP, Burlet-Schiltz O, Monsarrat B.
Journal: Methods Mol Biol (2008): 111
Page updated on September 19, 2024

Ordering information

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Unit size
Catalog Number13468
Quantity
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Physical properties

Molecular weight

1155.30

Solvent

DMSO

Spectral properties

Extinction coefficient (cm -1 M -1)

80000

Excitation (nm)

500

Emission (nm)

522

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateSolid black
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