Human Caspase-8 ELISA Kit (HUFI00491)- High Sensitivity

Product Name:	Human Caspase-8 ELISA Kit
Product Code:	HUFI00491
Size:	96 Assays
Alias:	CASP8, Caspase 8, Mch5, AIS, androgen receptor, DHTRTFM, HUMARA, NR3C4KD
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human CASP8 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human CASP8 and the recovery rates were calculated by comparing the measured value to the expected amount of Human CASP8 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	91-103	97
EDTA plasma(n=5)	85-96	90
UFH plasma(n=5)	87-98	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CASP8 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	91-103%	87-98%	86-105%
EDTA plasma(n=5)	85-98%	82-101%	82-94%
UFH plasma(n=5)	83-100%	80-96%	83-99%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

Q14790

UniProt Protein Function:	CASP8: Most upstream protease of the activation cascade of caspases responsible for the TNFRSF6/FAS mediated and TNFRSF1A induced cell death. Binding to the adapter molecule FADD recruits it to either receptor. The resulting aggregate called death- inducing signaling complex (DISC) performs CASP8 proteolytic activation. The active dimeric enzyme is then liberated from the DISC and free to activate downstream apoptotic proteases. Proteolytic fragments of the N-terminal propeptide (termed CAP3, CAP5 and CAP6) are likely retained in the DISC. Cleaves and activates CASP3, CASP4, CASP6, CASP7, CASP9 and CASP10. May participate in the GZMB apoptotic pathways. Cleaves ADPRT. Hydrolyzes the small-molecule substrate, Ac-Asp-Glu-Val-Asp-\|-AMC. Likely target for the cowpox virus CRMA death inhibitory protein. Isoform 5, isoform 6, isoform 7 and isoform 8 lack the catalytic site and may interfere with the pro-apoptotic activity of the complex. Heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a 18 kDa (p18) and a 10 kDa (p10) subunit. Interacts with FADD, CFLAR and PEA15. Isoform 9 interacts at the endoplasmic reticulum with a complex containing BCAP31, BAP29, BCL2 and/or BCL2L1. Interacts with TNFAIP8L2. Interacts with human cytomegalovirus/HHV-5 protein vICA/UL36; this interaction inhibits CASP8 activation. Isoform 1, isoform 5 and isoform 7 are expressed in a wide variety of tissues. Highest expression in peripheral blood leukocytes, spleen, thymus and liver. Barely detectable in brain, testis and skeletal muscle. Belongs to the peptidase C14A family. 9 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Protease; EC 3.4.22.61; Apoptosis Chromosomal Location of Human Ortholog: 2q33-q34 Cellular Component: nucleoplasm; mitochondrial outer membrane; neuron projection; cytoskeleton; mitochondrion; Noc1p-Noc2p complex; cytoplasm; microtubule organizing center; CD95 death-inducing signaling complex; cytosol; lipid raft Molecular Function:peptidase activity; protein binding; protein heterodimerization activity; cysteine-type endopeptidase activity; ubiquitin protein ligase binding; death receptor binding; protein complex binding; tumor necrosis factor receptor binding; cysteine-type peptidase activity Biological Process: macrophage differentiation; T cell activation; viral reproduction; apoptosis; protein heterooligomerization; positive regulation of proteolysis; heart development; natural killer cell activation; response to lipopolysaccharide; toll-like receptor 3 signaling pathway; proteolysis; response to estradiol stimulus; response to antibiotic; proteolysis involved in cellular protein catabolic process; angiogenesis; positive regulation of macrophage differentiation; toll-like receptor 4 signaling pathway; cell structure disassembly during apoptosis; caspase activation; positive regulation of I-kappaB kinase/NF-kappaB cascade; B cell activation; MyD88-independent toll-like receptor signaling pathway; negative regulation of I-kappaB kinase/NF-kappaB cascade; response to ethanol; induction of apoptosis via death domain receptors; toll-like receptor signaling pathway; response to cobalt ion; innate immune response; response to cold; neural tube formation Disease: Caspase 8 Deficiency; Breast Cancer; Lung Cancer; Hepatocellular Carcinoma
NCBI Summary:	This gene encodes a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. Caspases exist as inactive proenzymes composed of a prodomain, a large protease subunit, and a small protease subunit. Activation of caspases requires proteolytic processing at conserved internal aspartic residues to generate a heterodimeric enzyme consisting of the large and small subunits. This protein is involved in the programmed cell death induced by Fas and various apoptotic stimuli. The N-terminal FADD-like death effector domain of this protein suggests that it may interact with Fas-interacting protein FADD. This protein was detected in the insoluble fraction of the affected brain region from Huntington disease patients but not in those from normal controls, which implicated the role in neurodegenerative diseases. Many alternatively spliced transcript variants encoding different isoforms have been described, although not all variants have had their full-length sequences determined. [provided by RefSeq, Jul 2008]
UniProt Code:	Q14790
NCBI GenInfo Identifier:	2493531
NCBI Gene ID:	841
NCBI Accession:	Q14790.1
UniProt Secondary Accession:	Q14790,O14676, Q14791, Q14792, Q14793, Q14794, Q14795 Q14796, Q15780, Q15806, Q53TT5, Q8TDI1,
UniProt Related Accession:	Q14790
Molecular Weight:
NCBI Full Name:	Caspase-8
NCBI Synonym Full Names:	caspase 8, apoptosis-related cysteine peptidase
NCBI Official Symbol:	CASP8Ã‚ Ã‚
NCBI Official Synonym Symbols:	CAP4; MACH; MCH5; FLICE; ALPS2B; Casp-8Ã‚ Ã‚
NCBI Protein Information:	caspase-8; FADD-like ICE; MACH-alpha-1/2/3 protein; apoptotic protease Mch-5; MACH-beta-1/2/3/4 protein; apoptotic cysteine protease; ICE-like apoptotic protease 5; MORT1-associated ced-3 homolog; FADD-homologous ICE/CED-3-like protease; caspase 8, apoptosis-related cysteine protease
UniProt Protein Name:	Caspase-8
UniProt Synonym Protein Names:	Apoptotic cysteine protease; Apoptotic protease Mch-5; CAP4; FADD-homologous ICE/ced-3-like protease; FADD-like ICE; FLICE; ICE-like apoptotic protease 5; MORT1-associated ced-3 homolog
Protein Family:	Caspase
UniProt Gene Name:	CASP8Ã‚ Ã‚
UniProt Entry Name:	CASP8_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 Ã‚µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 Ã‚µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.