Mouse CASP4 (Caspase 4) ELISA Kit (MOES00812)

SKU:
MOES00812
€549

Description

ELISA Kit Technical ManualMSDS

Mouse CASP4 (Caspase 4) ELISA Kit

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 CASP4 . 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 CASP4 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 CASP4, 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 CASP4. The concentration of Mouse CASP4 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityMouse
Detection MethodColormetric
Detection Range1.56-100 ng/mL
Sensitivity0.94 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Mouse CASP4 in samples. No significant cross-reactivity or interference between Mouse CASP4 and analogues was observed.

Typical Data

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
1002.383
2.427
2.4052.323
501.658
1.66
1.6591.577
250.958
0.946
0.9520.87
12.50.425
0.445
0.4350.353
6.250.263
0.249
0.2560.174
3.130.187
0.175
0.1810.099
1.560.126
0.14
0.1330.051
00.079
0.085
0.082--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse CASP4 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse CASP4 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)4.7113.3739.284.5914.3139.94
Standard deviation0.280.771.360.230.711.36
C V (%)5.945.763.465.014.963.41

Recovery

The recovery of Mouse CASP4 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)86-10193
EDTA plasma (n=5)85-9890
Cell culture media (n=5)87-10093

Linearity

Samples were spiked with high concentrations of Mouse CASP4 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)
1:2Range (%)97-10893-10789-105
Average (%)10310197
1:4Range (%)93-10581-9389-103
Average (%)998895
1:8Range (%)93-10584-9981-96
Average (%)999088
1:16Range (%)89-10280-9089-99
Average (%)968594

Kit Components & Storage

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 X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Mouse CASP4 (Caspase 4) ELISA Kit (MOES00812) Assay procedure

    1. 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.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. 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.
    7. 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.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. 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.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Mouse CASP4 (Caspase 4) ELISA Kit (MOES00812) Protein Information

UniProt Protein Function:CASP4: Involved in the activation cascade of caspases responsible for apoptosis execution. Cleaves caspase-1. Heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a small and a large subunit. Widely expressed, with highest levels in spleen and lung. Moderate expression in heart and liver, low expression in skeletal muscle, kidney and testis. Not found in the brain. Belongs to the peptidase C14A family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:EC 3.4.22.64; Apoptosis; Protease

Cellular Component: cell soma; endoplasmic reticulum; membrane; mitochondrion; neuron projection; protein complex

Molecular Function:cysteine-type endopeptidase activity; cysteine-type peptidase activity; hydrolase activity; peptidase activity

Biological Process: actin filament organization; apoptosis; germ cell programmed cell death; positive regulation of interleukin-1 beta secretion; proteolysis; regulation of apoptosis; regulation of inflammatory response

NCBI Summary:This gene encodes a member of the cysteine proteases that plays important roles in apoptosis, cell migration and the inflammatory response. The encoded protein mediates production of pro-inflammatory cytokines by macrophages upon bacterial infection. Mice lacking the encoded protein are resistant to endotoxic shock induced by lipopolysaccharide. A 5-bp deletion encompassing a splice acceptor junction resulting in alternate splicing and a shorter non-functional isoform in certain mouse strains has been described. Although its official nomenclature is "caspase 4, apoptosis-related cysteine peptidase", this gene and its encoded protein have historically been called caspase 11. This gene is present in a cluster of three caspase genes on chromosome 9. [provided by RefSeq, Apr 2015]
UniProt Code:P70343
NCBI GenInfo Identifier:341940314
NCBI Gene ID:12363
NCBI Accession:P70343.2
UniProt Secondary Accession:P70343,O08735, Q3TAF3,
UniProt Related Accession:P70343
Molecular Weight:42,742 Da
NCBI Full Name:Caspase-4
NCBI Synonym Full Names:caspase 4, apoptosis-related cysteine peptidase
NCBI Official Symbol:Casp4
NCBI Official Synonym Symbols:Caspl; ich-3; CASP-4; Casp11; CASP-11
NCBI Protein Information:caspase-4
UniProt Protein Name:Caspase-4
UniProt Synonym Protein Names:Caspase-11; CASP-11
Protein Family:Caspase
UniProt Gene Name:Casp4
UniProt Entry Name:CASP4_MOUSE
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Additional Information

Product type:
ELISA
Reactivity:
Mouse
ELISA Type:
Sandwich
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