Product Name:	Mouse IL-1B ELISA Kit
Product Code:	MOFI00058
Size:	96 Assays
Alias:	IL-1beta, Interleukin 1 Beta, IL-1beta, IL1B, IL1-BETA, IL1F2, catabolin
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse IL-1beta concentrations in serum plasma and other biological fluids.
Sensitivity:	7.5pg/ml
Range:	12.5-800pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Mouse IL-1beta and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse IL-1beta in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-105	98
EDTA plasma(n=5)	89-101	94
UFH plasma(n=5)	87-102	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse IL-1beta 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%	89-105%	85-103%
EDTA plasma(n=5)	84-97%	90-99%	84-101%
UFH plasma(n=5)	80-97%	85-93%	83-99%

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

P10749

UniProt Protein Function:	IL1B: Produced by activated macrophages, IL-1 stimulates thymocyte proliferation by inducing IL-2 release, B-cell maturation and proliferation, and fibroblast growth factor activity. IL-1 proteins are involved in the inflammatory response, being identified as endogenous pyrogens, and are reported to stimulate the release of prostaglandin and collagenase from synovial cells. Monomer. Belongs to the IL-1 family.
UniProt Protein Details:	Protein type:Cytokine Cellular Component: extracellular space; cell; extracellular region; intracellular; cytosol; vesicle; secretory granule Molecular Function:protein domain specific binding; interleukin-1 receptor binding; cytokine activity; receptor binding Biological Process: positive regulation of granulocyte macrophage colony-stimulating factor production; positive regulation of JNK activity; negative regulation of MAP kinase activity; positive regulation of nitric oxide biosynthetic process; negative regulation of glutamate secretion; activation of MAPK activity; positive regulation of apoptosis; positive regulation of transcription, DNA-dependent; positive regulation of interleukin-2 biosynthetic process; germ cell programmed cell death; negative regulation of insulin receptor signaling pathway; positive regulation of glial cell differentiation; positive regulation of lipid catabolic process; positive regulation of NF-kappaB import into nucleus; response to lipopolysaccharide; fever; positive regulation of membrane protein ectodomain proteolysis; response to carbohydrate stimulus; activation of NF-kappaB transcription factor; elevation of cytosolic calcium ion concentration; positive regulation of phagocytosis; positive regulation of T cell proliferation; positive regulation of astrocyte differentiation; neutrophil chemotaxis; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of heterotypic cell-cell adhesion; positive regulation of mitosis; positive regulation of interleukin-6 production; interleukin-1 beta production; social behavior; positive regulation of angiogenesis; negative regulation of neuron differentiation; positive regulation of cell division; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; negative regulation of lipid metabolic process; leukocyte migration; sequestering of triacylglycerol; positive regulation of interleukin-6 biosynthetic process; positive regulation of JNK cascade; negative regulation of transcription from RNA polymerase II promoter; positive regulation of stress-activated MAPK cascade; negative regulation of neurogenesis; positive regulation of interleukin-8 production; negative regulation of cell proliferation; negative regulation of lipid catabolic process; hyaluronan biosynthetic process; lipopolysaccharide-mediated signaling pathway; protein kinase B signaling cascade; regulation of I-kappaB kinase/NF-kappaB cascade; inflammatory response; aging; cytokine and chemokine mediated signaling pathway; MAPKKK cascade; positive regulation of immature T cell proliferation in the thymus; response to ATP; memory; positive regulation of interferon-gamma production; positive regulation of chemokine biosynthetic process; positive regulation of prostaglandin secretion; positive regulation of fever; immune response; positive regulation of protein amino acid phosphorylation; regulation of insulin secretion
NCBI Summary:	The protein encoded by this gene is a member of the interleukin 1 cytokine family. This cytokine is produced by activated macrophages as a proprotein, which is proteolytically processed to its active form by caspase 1. The encoded protein plays a role in thymocyte proliferation and is involved in the inflammatory response. [provided by RefSeq, Aug 2015]
UniProt Code:	P10749
NCBI GenInfo Identifier:	124304
NCBI Gene ID:	16176
NCBI Accession:	P10749.1
UniProt Secondary Accession:	P10749,Q2M4J6,
UniProt Related Accession:	P10749
Molecular Weight:	30,931 Da
NCBI Full Name:	Interleukin-1 beta
NCBI Synonym Full Names:	interleukin 1 beta
NCBI Official Symbol:	Il1b
NCBI Official Synonym Symbols:	Il-1b; IL-1beta
NCBI Protein Information:	interleukin-1 beta; IL-1 beta
UniProt Protein Name:	Interleukin-1 beta
Protein Family:	Interleukin
UniProt Gene Name:	Il1b
UniProt Entry Name:	IL1B_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.

Antibodies	ELISA
NEU3 Antibody, Biotin conjugated	Mouse IL-1 beta CLIA Kit
Il1b Antibody	Mouse IL-1B ELISA Kit
Il1b Antibody, HRP conjugated
Il1b Antibody, FITC conjugated