Caspase-1 Fluorometric Assay Kit

Antigen: Caspase 1, Apoptosis-Related Cysteine Peptidase (Interleukin 1, Beta, Convertase) (CASP1)

Synonyms: ICE, P45, IL1BC, Il1bc, Ice, DCP-1, DCP1, l(2)01862, l(2)02132, DmelCG5370, CG5370, CASP1, LOC100033888

Application: Enzyme Activity Assay (EAA)

Catalog no.: ABIN411797

Additional Information

Alternative name: Caspase-1

Description: 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 ( λ max = 400 nm), upon cleavage of the substrate by caspase-1 or related caspases, free AFC emits a yellow-green fluorescence ( λ max = 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.

Application Details

Protocol: General Considerations Aliquot enough 2X Reaction Buffer for the number of assays to be performed. Add DTT to the 2X Reaction Buffer immediately before use (10 mM final concentration: add 10 μ l of 1.0 M DTT stock per 1 ml of 2X Reaction Buffer). After thawing, store the Cell Lysis Buffer and 2X Reaction Buffer at 4 o C. Protect YVAD-AFC from light. B.. 1. Induce apoptosis or treat cells by desired method. Concurrently incubate a control culture without treatment.

Application Notes: Note: Active Caspase-1 (BioVision, Cat.# 1081-25) can be used as a positive control. 2. Pellet 1-5 x 10 6 cells or use 50-200 μ g cell lysates if protein concentration has been measured. 3. Resuspend cells in 50 μ l of chilled Cell Lysis Buffer. Incubate cells on ice for 10 minutes. 4. Add 50 μ l of 2X Reaction Buffer (containing 10 mM DTT) to each sample. Add 5 μ l of the 1 mM YVAD-AFC substrate (50 μ M final concentration). Incubate at 37 o C for 1-2 hour. 5. Read samples in a fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter. For a plate-reading set-up, transfer the samples to a 96-well plate. You may also perform the entire assay directly in a 96-well plate. Fold-increase in Caspase-1 activity can be determined by comparing the results of induced samples with the level of the untreated control.

Storage: Store kit at –20 o C (Store Cell Lysis Buffer and 2X Reaction Buffer at 4 o C after opening). All reagents are stable for 6 months under proper storage conditions. V. Related Products : Apoptosis Detection Kits & Reagents • Annexin V Kits & Bulk Reagents • Caspase Assay Kits & Reagents • Mitochondrial Apoptosis Kits & Reagents • Nuclear Apoptosis Kits & Reagents • Apoptosis Inducers and Apoptosis siRNA Vectors Cell Fractionation System • Mitochondria/Cytosol Fractionation Kit • Nuclear/Cytosol Fractionation Kit • Membrane Protein Extraction Kit • FractionPREP Fractionation System Cell Proliferation & Senescence • Quick Cell Proliferation Assay Kit • Senescence Detection Kit • High Throughput Apoptosis/Cell Viability Assay Kits • LDH-Cytotoxicity Assay Kit Cell Damage & Repair • HDAC & HAT Fluorometric & Colorimetric Assays & Screening Kits • DNA Damage Quantification Kit • Glutathione & Nitric Oxide Fluorometric & Colorimetric Assay Kits Signal Transduction • cAMP & cGMP Assay Kits • Akt & JNK Activity Assay Kits Adipocyte & Lipid Transfer • Recombinant Adiponectin, Survivin, & Leptin • CETP & PLTP Activity Assay & Drug Discovery Kits • Total Cholesterol Quantification Kit Molecular Biology & Reporter Assays • siRNA Vectors • Cloning Insert Quick Screening Kit • Mitochondrial & Genomic DNA Isolation Kits • 5 Minutes DNA Ligation Kit • 20 Minutes Gel Staining/Destaining Kit Growth Factors and Cytokines • Adiponectin/Resistin/Leptin and their Antibodies • Recombinant Protein A and Protein G • Recombinant Complement C5a • Recombinant Cytokines and Growth Factors Monoclonal and Polyclonal Antibodies • Tag (HA, His, GST, Myc) Antibodies • Phospho Antibodies • Cellular Marker Antibodies • Biotinylated Antibodies • And many more Components K110-25 K110-100 K110-200 K110-400 25 assays 100 assays 200 assays 400 assays Cell Lysis Buffer 25 ml 100 ml 100 ml 100 ml 2X Reaction Buffer 2 ml 4 x 2 ml 16 ml 32 ml YVAD-AFC (1mM) 125 μ l 0.5 ml 2 x 0.5 ml 2 x 1 ml DTT (1 M) 100 μ l 400 μ l 400 μ l 400 μ l

Restrictions: For Research Use only

Publications

Publications: Cheng, van de Veerdonk, Lenardon 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." in: Journal of leukocyte biology, 2011 (PubMed).