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Caspase-1 Fluorometric Assay Kit

based on 10 citations in multiple journalsCaspase-1 Fluorometric Assay Kit105 4
Convenient, Highly-Sensitive Assay
Catalog #: K110
SKU-Size Size Price Qty
K110-25 25 assays
$145.00
K110-100 100 assays
$395.00
K110-200 200 assays
$525.00
K110-400 400 assays
$835.00
More Sizes Get Quote

Product Details

Detection Method Fluorescence (400 nm excitation filter and 505 nm emission filter)
Species Reactivity Mammalian
Applications Detect early/middle stages of apoptosis; differentiate apoptosis from necrosis.
Features & Benefits • Simple one-step procedure; takes 1-2 hours
• Fast and convenient
• Comparison of the fluorescence of AFC from a treated sample with an untreated control allows determination of the fold increase in caspase-1 activity.
Kit Components • Cell Lysis Buffer
• 2X Reaction Buffer
• YVAD-AFC (1mM)
• DTT (1 M)
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.

Details

Activation of ICE-family proteases/caspases initiates apoptosis or other cellular processes in mammalian cells. The Caspase-1/ICE Fluorometric Assay Kit provides a simple and convenient means for assaying the activity of caspases that recognize the sequence YVAD. The assay is based on detection of cleavage of substrate YVAD-AFC (AFC: 7-amino-4-trifluoromethyl coumarin). YVAD-AFC emits blue light (400 nm); upon cleavage of the substrate by caspase-1 or related caspases, free AFC emits a yellow-green fluorescence (505 nm), which can be quantified using a fluorometer or a fluorecence microtiter plate reader. Comparison of the fluorescence of AFC from a treated sample with an untreated control allows determination of the fold increase in caspase-1 activity.


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Do the protease inhibitor that I added will interfere with the assay?
Protease inhibitors will interefere with the assay. Caspase 1 is a cysteine protease and can be inhibited by most protease inhibitor cocktails. The assay is based on the catalytic clevage of the substrate YVAD-AFC by Caspase-1. Inhibition of caspase 1 would thus interfere with the measurement.
Can you test plasma samples?
The kit is good for cell and tissue lysates only and not for plasma samples.
Is the substrate cell permeable?
This is an endpoint assay where cells are lysed and the assay is performed with cell lysate. So cell permeability is not an issue.
What is DTT for?
A reducing environment is essential for caspases to stay in solution and to be active. DTT helps in that process.
How should tissue lysates be prepared?
For tissues, homogenize 10 mg of tissue in 200 ul of lysis buffer. Spin out debris and add 50 ul of supernatant into a well of the 96-well plate then proceed with the protocol.
Min Guo, et al., (2018) Ketogenic Diet Improves Brain Ischemic Tolerance and Inhibits NLRP3 Inflammasome Activation by Preventing Drp1-Mediated Mitochondrial Fission and Endoplasmic Reticulum Stress, Frontiers in Molecular Neuroscience, Mar. 2018, 29662437
Ahn H et al., (2017) Isorhamnetin and hyperoside derived from water dropwort inhibits inflammasome activation, Phytomedicine, 2017, 24:77-86
Lim et al., Regiospecific Methylation of a Dietary Flavonoid Scaffold Selectively Enhances IL-1β Production following Toll-like Receptor 2 Stimulation in THP-1 Monocytes. J. Biol. Chem., Jul 2013; 288: 21126 - 21135.
Koenen, T. B. et. al. The Inflammasome and Caspase-1 Activation: A New Mechanism Underlying Increased Inflammatory Activity in Human Visceral Adipose Tissue. Endocrinology, Oct 2011; 152: 3769 - 3778.
Cheng, S. C. et. al. The dectin-1/inflammasome pathway is responsible for the induction of protective T-helper 17 responses that discriminate between yeasts and hyphae of Candida albicans. J. Leukoc. Biol., Aug 2011; 90: 357 - 366.
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