Mouse IL-12 p40 ELISA Kit (MOFI01217)- High Sensitivity

Product Name:	Mouse IL-12 p40 ELISA Kit (MOFI01217)
Product Code:	MOFI01217
Size:	96 Assays
Alias:	Interleukin 12 p40
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse IL-12 p40 concentrations in serum plasma and other biological fluids.
Sensitivity:	9.375pg/ml
Range:	15.625-1000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:	Matrices listed below were spiked with certain level of Mouse IL-12 p40 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse IL-12 p40 in samples. Not Available.
Linearity:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse IL-12 p40 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Not Available.
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

P43432

UniProt Protein Function:	IL12B: Cytokine that can act as a growth factor for activated T and NK cells, enhance the lytic activity of NK/lymphokine- activated killer cells, and stimulate the production of IFN-gamma by resting PBMC. Defects in IL12B are a cause of mendelian susceptibility to mycobacterial disease (MSMD); also known as familial disseminated atypical mycobacterial infection. This rare condition confers predisposition to illness caused by moderately virulent mycobacterial species, such as Bacillus Calmette-Guerin (BCG) vaccine and environmental non-tuberculous mycobacteria, and by the more virulent Mycobacterium tuberculosis. Other microorganisms rarely cause severe clinical disease in individuals with susceptibility to mycobacterial infections, with the exception of Salmonella which infects less than 50% of these individuals. The pathogenic mechanism underlying MSMD is the impairment of interferon-gamma mediated immunity, whose severity determines the clinical outcome. Some patients die of overwhelming mycobacterial disease with lepromatous-like lesions in early childhood, whereas others develop, later in life, disseminated but curable infections with tuberculoid granulomas. MSMD is a genetically heterogeneous disease with autosomal recessive, autosomal dominant or X-linked inheritance. Genetic variations in IL12B are a cause of susceptibility to psoriasis type 11 (PSORS11). Psoriasis is a common, chronic inflammatory disease of the skin with multifactorial etiology. It is characterized by red, scaly plaques usually found on the scalp, elbows and knees. These lesions are caused by abnormal keratinocyte proliferation and infiltration of inflammatory cells into the dermis and epidermis. Belongs to the type I cytokine receptor family. Type 3 subfamily.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Cytokine; Secreted Cellular Component: cytoplasm; extracellular space; interleukin-12 complex Molecular Function:cytokine activity; growth factor activity; hematopoietin/interferon-class (D200-domain) cytokine receptor binding; identical protein binding; interleukin-12 alpha subunit binding; interleukin-12 receptor binding; interleukin-23 receptor binding; protein binding; protein heterodimerization activity; protein homodimerization activity Biological Process: cell cycle arrest; cell migration; cell surface receptor linked signal transduction; defense response to Gram-negative bacterium; defense response to protozoan; defense response to virus; natural killer cell activation; natural killer cell activation during immune response; negative regulation of inflammatory response to antigenic stimulus; negative regulation of interleukin-10 production; negative regulation of interleukin-17 production; negative regulation of smooth muscle cell proliferation; positive regulation of activated T cell proliferation; positive regulation of cell adhesion; positive regulation of defense response to virus by host; positive regulation of granulocyte macrophage colony-stimulating factor production; positive regulation of interferon-gamma biosynthetic process; positive regulation of interferon-gamma production; positive regulation of interleukin-10 production; positive regulation of interleukin-12 production; positive regulation of interleukin-17 production; positive regulation of lymphocyte proliferation; positive regulation of mononuclear cell proliferation; positive regulation of natural killer cell activation; positive regulation of natural killer cell mediated cytotoxicity directed against tumor cell target; positive regulation of natural killer cell proliferation; positive regulation of NK T cell activation; positive regulation of NK T cell proliferation; positive regulation of osteoclast differentiation; positive regulation of T cell mediated cytotoxicity; positive regulation of T cell proliferation; positive regulation of T-helper 1 type immune response; positive regulation of tumor necrosis factor production; positive regulation of tyrosine phosphorylation of Stat3 protein; positive regulation of tyrosine phosphorylation of Stat4 protein; positive regulation of tyrosine phosphorylation of Stat5 protein; regulation of tyrosine phosphorylation of Stat1 protein; response to organic substance; response to UV-B; sensory perception of pain; T-helper cell differentiation
NCBI Summary:	This gene encodes the beta subunit p40 of the Interleukin 12 (IL-12) family of cytokines. Members of the IL-12 family form heterodimers consisting of heavy and light subunits linked by disulfide bonds. The product of this gene, p40, is a subunit of interleukins IL-12 and IL-23. [provided by RefSeq, Dec 2014]
UniProt Code:	P43432
NCBI GenInfo Identifier:	1170461
NCBI Gene ID:	16160
NCBI Accession:	P43432.1
UniProt Secondary Accession:	P43432,Q9QUM1,
UniProt Related Accession:	P43432
Molecular Weight:	38,235 Da
NCBI Full Name:	Interleukin-12 subunit beta
NCBI Synonym Full Names:	interleukin 12b
NCBI Official Symbol:	Il12b
NCBI Official Synonym Symbols:	p40; Il-12b; Il12p40; Il-12p40
NCBI Protein Information:	interleukin-12 subunit beta
UniProt Protein Name:	Interleukin-12 subunit beta
UniProt Synonym Protein Names:	Cytotoxic lymphocyte maturation factor 40 kDa subunit; CLMF p40; IL-12 subunit p40
Protein Family:	Interleukin
UniProt Gene Name:	Il12b
UniProt Entry Name:	IL12B_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.