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Caspase-1 Colorimetric Assay Kit

based on 8 citations in multiple journalsCaspase-1 Colorimetric Assay Kit84.2 4
Convenient, Highly-Sensitive Assay
Catalog #: K111
SKU-Size Size Price Qty
K111-25 25 assays
$145.00
K111-100 100 assays
$395.00
K111-200 200 assays
$525.00
K111-400 400 assays
$835.00
More Sizes Get Quote

Product Details

Detection Method Absorbance (400 or 405 nm)
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 absorbance of pNA 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-pNA (4 mM)
• DTT (1 M)
• Dilution Buffer
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 Colorimetric Protease Assay Kits provide a simple and convenient means for assaying the activity of caspases that recognize the sequence YVAD. The assay is based on spectrophotometric detection of the chromophore p-nitroanilide (pNA) after cleavage from the labeled substrate YVAD-pNA. The pNA light emission can be quantified using a spectrophotometer or a microtiter plate reader at 400- or 405 nm. Comparison of the absorbance of pNA 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-pNA 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.
We would like to detect caspase-1 activity in mouse brain tissue. How can we prepare the tissue homogenate? Should we lyse the intact tissue directly in cell lysis buffer provided with the kit or alternatively, can we use protein lysates already made with RIPA buffer?
The assay has been standardized after inducing apoptosis in cultured cells. You can use the cell lysis buffer provided with the kit for preparing tissue homogenates. RIPA may be too harsh on the enzymes and we do not recommend it. Once tissue is lysed in cell lysis buffer, centrifuge at 10,000g for 15 min and use the supernatant for the assay.
Maria C. Edman, et al., (2018) Increased Cathepsin S activity associated with decreased protease inhibitory capacity contributes to altered tear proteins in Sjögren’s Syndrome patients, Scientific Reports, Jul. 2018, 30038391
Marni E. Cueno, et al., (2018) Gingival Periodontal Disease (PD) Level-Butyric Acid Affects the Systemic Blood and Brain Organ: Insights Into the Systemic Inflammation of Periodontal Disease, Frontiers in Microbiology, Jun. 2018, 29915575
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.
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