Proteasomal Chymotrypsin-like Activity Assay

Purified rabbit 20S proteasome (35 ng) was incubated with 40 μmol/L of fluorogenic peptide substrate Suc-LLVY-AMC (for the proteasomal chymotrypsin-like activities) in 100 μl assay buffer (20 mM Tris-HCl, pH 7.5) in the presence of shikonin at different concentrations or the solvent DMSO for 2 h at 37°C, followed by measurement of hydrolysis of the fluorogenic substrates using a Wallac Victor3™ multilabel counter with 355-nm excitation and 460-nm emission wavelengths.

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Yang H., Zhou P., Huang H., Chen D., Ma N., Cui C.Q., Shen S., Dong W., Zhang X., Lian W., Wang X., Dou Q.P, & Liu J. (2009). Shikonin Exerts Antitumor Activity via Proteasome Inhibition and Cell Death Induction in vitro and in vivo. International journal of cancer. Journal international du cancer, 124(10), 2450-2459.

Publication 2009

Assay Buffer Chymotrypsin Dmso Fluorogenic substrates Hydrolysis Peptide l Proteasomal Rabbit Shikonin Solvent Tris

Corresponding Organization :

Other organizations : Guangzhou Medical University, The Barbara Ann Karmanos Cancer Institute, Wayne State University, University of South Dakota

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Variable analysis

independent variables

Shikonin at different concentrations

dependent variables

Hydrolysis of the fluorogenic substrates (Suc-LLVY-AMC)

control variables

Purified rabbit 20S proteasome (35 ng)
Suc-LLVY-AMC (40 μmol/L)
Assay buffer (20 mM Tris-HCl, pH 7.5)
Incubation time (2 h)
Temperature (37°C)
DMSO (solvent control)

controls

Positive control: Purified rabbit 20S proteasome incubated with Suc-LLVY-AMC in the absence of shikonin (DMSO control)
Negative control: Not explicitly mentioned

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