Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
Detection Method:	Colormetric
Detection Range:	0.16-10 ng/mL
Sensitivity:	0.10 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Mouse CASP3 in samples. No significant cross-reactivity or interference between Mouse CASP3 and analogues was observed.

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Mouse CASP3. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse CASP3 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Mouse CASP3, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Mouse CASP3. The concentration of Mouse CASP3 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	CASP3: Involved in the activation cascade of caspases responsible for apoptosis execution. At the onset of apoptosis it proteolytically cleaves poly(ADP-ribose) polymerase (PARP) at a '216-Asp-\|-Gly-217' bond. Cleaves and activates sterol regulatory element binding proteins (SREBPs) between the basic helix-loop- helix leucine zipper domain and the membrane attachment domain. Cleaves and activates caspase-6, -7 and -9. Involved in the cleavage of huntingtin. Triggers cell adhesion in sympathetic neurons through RET cleavage. Heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a 17 kDa (p17) and a 12 kDa (p12) subunit. Interacts with BIRC6/bruce. Highly expressed in lung, spleen, heart, liver and kidney. Moderate levels in brain and skeletal muscle, and low in testis. Also found in many cell lines, highest expression in cells of the immune system. Inhibited by isatin sulfonamides. Belongs to the peptidase C14A family.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Protease; Apoptosis; EC 3. 4. 22. 56 Cellular Component: cytoplasm; cytosol; lipid raft; nucleus Molecular Function:aspartic-type endopeptidase activity; cyclin-dependent protein kinase inhibitor activity; cysteine-type endopeptidase activity; cysteine-type peptidase activity; death receptor binding; peptidase activity; phospholipase A2 activator activity; protease binding; protein binding; protein complex binding Biological Process: apoptosis; B cell homeostasis; cell fate commitment; erythrocyte differentiation; heart development; keratinocyte differentiation; learning and/or memory; negative regulation of activated T cell proliferation; negative regulation of apoptosis; negative regulation of B cell proliferation; negative regulation of cell cycle; negative regulation of cyclin-dependent protein kinase activity; nerve growth factor receptor signaling pathway; neuron apoptosis; neuron differentiation; positive regulation of apoptosis; positive regulation of neuron apoptosis; protein processing; proteolysis; response to DNA damage stimulus; response to glucose stimulus; response to hydrogen peroxide; response to organic substance; response to UV; response to wounding; sensory perception of sound; T cell homeostasis
NCBI Summary:	This gene encodes a protein that belongs to a highly conserved family of cysteinyl aspartate-specific proteases that function as essential regulators of programmed cell death through apoptosis. Members of this family contain an N-terminal pro-domain and require cleavage at specific aspartate residues to become mature. The protein encoded by this gene belongs to a subgroup of cysteinyl aspartate-specific proteases that are activated by initiator caspases and that perform the proteolytic cleavage of apoptotic target proteins. Mice defective for this gene exhibit a variety of phenotypes including reduced neuronal apoptosis resulting in hyperplasias, hearing loss, attenuated osteogenic differentiation of bone marrow stromal stem cells, and pre- and post-natal lethality. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Sep 2015]
UniProt Code:	P70677
NCBI GenInfo Identifier:	2493527
NCBI Gene ID:	12367
NCBI Accession:	P70677. 1
UniProt Secondary Accession:	P70677,O08668, Q8CHV5, Q9QWI4,
UniProt Related Accession:	P70677
Molecular Weight:	31,475 Da
NCBI Full Name:	Caspase-3
NCBI Synonym Full Names:	caspase 3
NCBI Official Symbol:	Casp3
NCBI Official Synonym Symbols:	CC3; AC-3; Lice; Yama; mldy; CPP32; SCA-1; CASP-3; CPP-32; A830040C14Rik
NCBI Protein Information:	caspase-3
UniProt Protein Name:	Caspase-3
UniProt Synonym Protein Names:	Apopain; Cysteine protease CPP32; CPP-32
Protein Family:	Caspase
UniProt Gene Name:	Casp3
UniProt Entry Name:	CASP3_MOUSE

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (ng/mL)	O.D	Average	Corrected
10	2.397 2.437	2.417	2.328
5	1.677 1.723	1.7	1.611
2.5	1.004 1	1.002	0.913
1.25	0.555 0.559	0.557	0.468
0.63	0.322 0.294	0.308	0.219
0.32	0.205 0.187	0.196	0.107
0.16	0.134 0.152	0.143	0.054
0	0.082 0.096	0.089	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse CASP3 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse CASP3 were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/mL)	0.53	1.29	4.47	0.50	1.40	4.20
Standard deviation	0.03	0.06	0.19	0.03	0.06	0.23
C V (%)	5.66	4.65	4.25	6.00	4.29	5.48

Recovery

The recovery of Mouse CASP3 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	86-97	92
EDTA plasma (n=5)	91-105	96
Cell culture media (n=5)	85-101	92

Linearity

Samples were spiked with high concentrations of Mouse CASP3 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	90-101	97-113	90-104
1:2	Average (%)	95	104	97
1:4	Range (%)	88-101	80-91	82-95
1:4	Average (%)	95	85	89
1:8	Range (%)	91-106	87-98	85-97
1:8	Average (%)	98	92	91
1:16	Range (%)	87-100	81-94	84-96
1:16	Average (%)	92	87	90

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro ELISA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.