Caspase-3, 7 Activity Fluorometric Assay Kit

Caspase-3, 7 Activity Fluorometric Assay Kit can be used for assaying caspase-3, 7 activities in cell/tissue extracts in a 96-well plate format. Each of the supplied fluorogenic substrate and the pan caspase inhibitor Z-VAD-FMK is sufficient for 250 x 100 µl reactions. The supplied TRAIL-treated cell lysate is used as a positive control, sufficient for 5 X 100 µl reactions.

Additional Information

Product Name: Caspase-3, 7 Activity Fluorometric Assay Kit
Also Known As: N/A
Catalog No.: J7130
Size: 1 Kit (250 assays)
Molecular Weight: N/A
Species: N/A
Source: N/A
Stock: N/A
Concentration: See tube label
Quality Assurance: Inhibitor and substrates > 98% by HPLC and NMR
Storage: Store at -80°C; avoid multiple freeze-thaw cycles
PDF Data Sheet: PDF datasheet, MSDS
NCBI RefSeq: N/A
Image(s): J7130 image


HCT116 cells were treated with 50 ng/ml 6xHis-TRAIL for 4 hours to activate extrinsic apoptosis. 25 μg cell lysates were incubated with (open circles) or without (solid circles) 20 μM Z-VAD-FMK (a pan caspase inhibitor) for 10 min at 37 0C. 25 μM Ac-LEHD-AFC (A) or Ac-DEVD-AFC (B) was added to initiate the reaction. AFC fluorescence was recorded with a plate reader using excitation/emission filter set at 400±40/508±30 nm, respectively.

Shipping Method: Dry ice shipping
References: N/A

Details

Caspase-3, 7 Activity Fluorometric Assay Kit can be used for assaying caspase-3, 7 activities in cell/tissue extracts in a 96-well plate format. Caspase-3, 7 cleave the supplied fluorogenic substrate. The fluorescence of released AFC can be monitored using a plate reader or a fluorometer at the excitation/emission wavelength of 400 nm/505 nm, respectively. The 96-well format assay can be completed in 2-4 hours.

Ac-DEVD-AFC is a fluorogenic substrate of caspase-3/7. Z-VAD-FMK is a pan caspase inhibitor, used to inhibit caspases in reactions to obtain background signals. TRAIL-treated HCT116 cell lysates contain activated caspase-8/9/3/7, used as a positive control. AFC is used for generating a standard curve for quantitating the amount of AFC produced in reactions.

Components

• 1000X Ac-DEVD-AFC (25 mM in DMSO)..................25 µl
• 1000X Z-VAD-FMK (25 mM in DMSO).......................25 µl
• TRAIL-treated HCT116 cell lysates (5 mg/ml)............25 µl
• AFC (0.1 mM in DMSO)..............................................250 µl
• Tris (2 M, pH 7.1 at 37°C) ..........................................1 ml
• DTT (200 mM).............................................................1 ml