CASP4 ELISA Kit (Human)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level Caspase 4 (CASP4) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level Caspase 4 (CASP4) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

Apoptotic cysteine protease Mih1/TX; CASP-4; caspase 4, apoptosis-related cysteine peptidase; caspase 4, apoptosis-related cysteine protease; caspase-4; caspase-4 isoform alpha; ICE and Ced-3 homolog 2; ICE (rel) II; ICE (rel) -II; ICEREL-II; ICH-2; Mih1; Mih1/TX; protease ICH-2; protease TX; TX.

Inflammatory caspase (PubMed:7797510, PubMed:23516580, PubMed:25119034) . Essential effector of NLRP3 inflammasome-dependent CASP1 activation and IL1B and IL18 secretion in response to non-canonical activators, such as UVB radiation, cholera enterotoxin subunit B and cytosolic LPS (PubMed:22246630, PubMed:26174085, PubMed:26173988, PubMed:26508369, PubMed:25964352) . Independently of NLRP3 inflammasome and CASP1, promotes pyroptosis, through GSDMD cleavage and activation, and IL1A, IL18 and HMGB1 release in response to non-canonical inflammasome activators (PubMed:24879791, PubMed:25964352) . Plays a crucial role in the restriction of Salmonella typhimurium replication in colonic epithelial cells during infection (PubMed:25121752) . In later stages of the infection, LPS from cytosolic Salmonella triggers CASP4 activation, which ultimately results in pyroptosis of infected cells and their extrusion into the gut lumen, as well as in IL18 secretion. Pyroptosis limits bacterial replication, while cytokine secretion promotes the recruitment and activation of immune cells and triggers mucosal inflammation. Involved in LPS-induced IL6 secretion; this activity may not require caspase enzymatic activity (PubMed:26508369) . Involved in cell death induced by endoplasmic reticulum stress and by treatment with cytotoxic APP peptides found Alzheimer's patient brains (PubMed:15123740, PubMed:22246630, PubMed:23661706) . Activated by direct binding to LPS without the need of an upstream sensor (PubMed:25119034) . Does not directly process IL1B (PubMed:7743998, PubMed:7797592, PubMed:7797510) . During non-canonical inflammasome activation, cuts CGAS and may play a role in the regulation of antiviral innate immune activation (PubMed:28314590) .

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Caspase 4 (CASP4) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Caspase 4 (CASP4) . Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Caspase 4 (CASP4), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm +/- 10nm. The concentration of Caspase 4 (CASP4) in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

Component Amount Anti-CASP4 Microplate 96 Wells (12 x 8 Well strips) CASP4 Lyophilized Standard 2 100X Biotinylated CASP4 Detector Antibody 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 120 uL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL Stop Solution 1 x 6 mL TMB Substrate 1 x 9 mL Component Amount Component Amount Anti-CASP4 Microplate 96 Wells (12 x 8 Well strips) Anti-CASP4 Microplate 96 Wells (12 x 8 Well strips) CASP4 Lyophilized Standard 2 CASP4 Lyophilized Standard 2 100X Biotinylated CASP4 Detector Antibody 1 x 120 uL 100X Biotinylated CASP4 Detector Antibody 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 120 uL Standard Diluent 1 x 20 mL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL 30X Wash Buffer 1 x 20 mL Stop Solution 1 x 6 mL Stop Solution 1 x 6 mL TMB Substrate 1 x 9 mL TMB Substrate 1 x 9 mL

This assay has high sensitivity and excellent specificity for detection of Caspase 4 (CASP4) . No significant cross-reactivity or interference between Caspase 4 (CASP4) and analogues was observed.