Boc-Leu-Arg-Arg-AMC (Boc-LRR-AMC)

Boc-LRR-AMC is a fluorogenic substrate for the trypsin – like activity of the 20S proteasome. Working concentrations of this substrate is 50-200 µM. The released AMC can be detected by a fluorimeter or plate reader at excitation/emission wavelengths of 380 nm/460 nm, respectively.

When used to determine proteasome activity in cell lysates, cell lysates that are pre-treated with a proteasome inhibitor such as MG132, PS341 or epoxomicin can be used to determine the fluorescence contributed by other cellular proteases that cleave this substrate. Readings from proteasome inhibitor-treated lysates should be subtracted.

Additional Information

Product Name: Boc-Leu-Arg-Arg-AMC (Boc-LRR-AMC)
Also Known As: Boc-LRR-AMC
Catalog No.: G3100
Size: 5 mg
Molecular Weight: 773.8 Da
Species: N/A
Source: Synthetic
Stock: Powder
Concentration: N/A
Quality Assurance: >98% by HPLC and NMR
Storage: Eligible for room temperature shipping. Store at -20°C upon receiving; avoid multiple freeze-thaw cycles after dissolving in DMSO.
PDF Data Sheet: PDF datasheet, MSDS
NCBI RefSeq: N/A
Image(s): G3100 G3101 image

20 nM constitutive (catalog # A1400) or immuno (catalog # A1500) bovine 20S proteasome was incubated with 120 nM PA28beta (catalog # A2200) for 15 min in 20 mM Tris, pH 7.1 at 37 0C, 50 mM NaCl, 2 mM bME . Each substrate was prepared in the same buffer at 100 µM. Then 50 µl constitutive or immune 20S proteasome was mixed with 50 µl each of the substrates into a well of a 96-well plate, and AMC fluorescence was recorded immediately in a 20 min kinetic mode using the exciting/emission filter set at 360/460 nm, respectively. Linear slop of each curve was used to represent the 20S proteasome activity. Values from substrate alone were substracted as background. Error bars represent S.D. of three assays.
Shipping Method: Room temperature shipping
References: 1. Chen P, et al. (1996) Cell 86(6), 961 – 972.
2. Arendt CS, et al. (1997) Proc Natl Acad Sci 94(14), 7156 – 7161.
3. Reidlinger J, et al. (1997) J Biol Chem 272(40), 24899 – 24905.

Details

Boc-LRR-AMC is a fluorogenic substrate for the trypsin – like activity of the 20S proteasome. Working concentrations of this substrate is 50-200 µM. The released AMC can be detected by a fluorimeter or plate reader at excitation/emission wavelengths of 380 nm/460 nm, respectively. When used to determine proteasome activity in cell lysates, cell lysates that are pre-treated with a proteasome inhibitor such as MG132, PS341 or epoxomicin can be used to determine the fluorescence contributed by other cellular proteases that cleave this substrate. Readings from proteasome inhibitor-treated lysates should be subtracted.