Product Name:	Mouse Caspase 1 ELISA Kit
Product Code:	MOFI00692
Size:	96 Assays
Alias:	CASP1, Caspase-1, ICE, CASP-1, ICEP45, IL-1 beta-converting enzyme, IL-1BC, IL1BCCASP1 nirs variant 1, IL1BCE, IL1B-convertase, interleukin 1-B converting enzyme, interleukin 1-beta convertase
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse CASP1 concentrations in serum plasma and other biological fluids.
Sensitivity:	18.75pg/ml
Range:	31.25-2000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Mouse CASP1 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse CASP1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	91-104	97
EDTA plasma(n=5)	90-99	94
UFH plasma(n=5)	86-105	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse CASP1 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)	89-102%	85-105%	96-104%
EDTA plasma(n=5)	84-101%	84-97%	82-95%
UFH plasma(n=5)	84-98%	86-99%	80-100%

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

P29452

UniProt Protein Function:	CASP1: Thiol protease that cleaves IL-1 beta between an Asp and an Ala, releasing the mature cytokine which is involved in a variety of inflammatory processes. Important for defense against pathogens. Cleaves and activates sterol regulatory element binding proteins (SREBPs). Can also promote apoptosis. Heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a 20 kDa (p20) and a 10 kDa (p10) subunit. The p20 subunit can also form a heterodimer with the epsilon isoform which then has an inhibitory effect. May be a component of the inflammasome, a protein complex which also includes PYCARD, CARD8 and NALP2 and whose function would be the activation of proinflammatory caspases. Interacts with CARD17/INCA and CARD18. Expressed in larger amounts in spleen and lung. Detected in liver, heart, small intestine, colon, thymus, prostate, skeletal muscle, peripheral blood leukocytes, kidney and testis. No expression in the brain. Specifically inhibited by the cowpox virus Crma protein. Belongs to the peptidase C14A family. 5 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Apoptosis; EC 3.4.22.36; Protease Cellular Component: neuron projection; protein complex; mitochondrion; cytoplasm; extracellular region; nucleus Molecular Function:peptidase activity; protein binding; hydrolase activity; endopeptidase activity; cysteine-type peptidase activity Biological Process: response to drug; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of apoptosis; apoptosis; membrane hyperpolarization; microglial cell activation; interleukin-1 beta production; response to lipopolysaccharide; mitochondrial depolarization; positive regulation of interleukin-1 beta secretion; proteolysis; response to organic cyclic substance; memory; response to ATP; positive regulation of interleukin-1 alpha secretion; regulation of apoptosis; response to bacterium; regulation of inflammatory response; response to hypoxia; protein processing; regulation of autophagy; positive regulation of circadian sleep/wake cycle, non-REM sleep; myoblast fusion; positive regulation of cytokine secretion
UniProt Code:	P29452
NCBI GenInfo Identifier:	266322
NCBI Gene ID:	12362
NCBI Accession:	P29452.1
UniProt Related Accession:	P29452
Molecular Weight:
NCBI Full Name:	Caspase-1
NCBI Synonym Full Names:	caspase 1
NCBI Official Symbol:	Casp1
NCBI Official Synonym Symbols:	ICE; Il1bc
NCBI Protein Information:	caspase-1; p45; CASP-1; IL-1BC; IL-1B converting enzyme; IL-1 beta-converting enzyme; interleukin-1 beta convertase; interleukin 1 beta-converting enzyme; interleukin-1 beta-converting enzyme
UniProt Protein Name:	Caspase-1
UniProt Synonym Protein Names:	Interleukin-1 beta convertase; IL-1BC; Interleukin-1 beta-converting enzyme; ICE; IL-1 beta-converting enzyme; p45Cleaved into the following 2 chains:Caspase-1 subunit p20; Caspase-1 subunit p10
UniProt Gene Name:	Casp1
UniProt Entry Name:	CASP1_MOUSE

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

Step	Procedure
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 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 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.