Human IL-12B ELISA Kit (HUEB0018)
- SKU:
- HUEB0018
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P29460
- Range:
- 31.2-2000 pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- IL-12, p40, IL12B, IL-12B, CLMF2, NKSF2
- Reactivity:
- Human
Description
| Product Name: | Human IL-12B ELISA Kit |
| Product Code: | HUEB0018 |
| Alias: | Interleukin-12 subunit beta, IL-12B, Cytotoxic lymphocyte maturation factor 40 kDa subunit, CLMF p40, IL-12 subunit p40, NK cell stimulatory factor chain 2, NKSF2, IL12B, NKSF2 |
| Uniprot: | P29460 |
| Reactivity: | Human |
| Range: | 31.2-2000 pg/mL |
| Detection Method: | Sandwich |
| Size: | 96 Assay |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| 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; Cytokine; Secreted, signal peptide Chromosomal Location of Human Ortholog: 5q31.1-q33.1 Cellular Component: extracellular space; membrane; interleukin-12 complex; cytoplasm Molecular Function:hematopoietin/interferon-class (D200-domain) cytokine receptor activity; identical protein binding; interleukin-12 alpha subunit binding; protein binding; protein homodimerization activity; growth factor activity; protein heterodimerization activity; interleukin-23 receptor binding; cytokine activity; interleukin-12 receptor binding Biological Process: positive regulation of granulocyte macrophage colony-stimulating factor production; positive regulation of cell adhesion; positive regulation of T-helper 1 type immune response; negative regulation of interleukin-10 production; positive regulation of T cell mediated cytotoxicity; negative regulation of smooth muscle cell proliferation; positive regulation of interleukin-12 production; positive regulation of osteoclast differentiation; positive regulation of NK T cell proliferation; positive regulation of tyrosine phosphorylation of Stat4 protein; natural killer cell activation; positive regulation of NF-kappaB import into nucleus; sensory perception of pain; defense response to Gram-negative bacterium; positive regulation of activated T cell proliferation; positive regulation of interleukin-10 production; positive regulation of natural killer cell activation; positive regulation of tyrosine phosphorylation of Stat3 protein; positive regulation of lymphocyte proliferation; natural killer cell activation during immune response; positive regulation of T cell proliferation; negative regulation of inflammatory response to antigenic stimulus; cell cycle arrest; defense response to virus; regulation of cytokine biosynthetic process; positive regulation of memory T cell differentiation; positive regulation of interleukin-17 production; positive regulation of natural killer cell proliferation; cell migration; T-helper 1 type immune response; positive regulation of NK T cell activation; T-helper cell differentiation; positive regulation of natural killer cell mediated cytotoxicity directed against tumor cell target; cytokine and chemokine mediated signaling pathway; defense response to protozoan; positive regulation of tumor necrosis factor production; regulation of tyrosine phosphorylation of Stat1 protein; response to UV-B; positive regulation of tissue remodeling; positive regulation of interferon-gamma production; negative regulation of interleukin-17 production; positive regulation of mononuclear cell proliferation; positive regulation of tyrosine phosphorylation of Stat5 protein; sexual reproduction; interferon-gamma biosynthetic process; positive regulation of defense response to virus by host; positive regulation of inflammatory response; positive regulation of interferon-gamma biosynthetic process Disease: Immunodeficiency 29 |
| NCBI Summary: | This gene encodes a subunit of interleukin 12, a cytokine that acts on T and natural killer cells, and has a broad array of biological activities. Interleukin 12 is a disulfide-linked heterodimer composed of the 40 kD cytokine receptor like subunit encoded by this gene, and a 35 kD subunit encoded by IL12A. This cytokine is expressed by activated macrophages that serve as an essential inducer of Th1 cells development. This cytokine has been found to be important for sustaining a sufficient number of memory/effector Th1 cells to mediate long-term protection to an intracellular pathogen. Overexpression of this gene was observed in the central nervous system of patients with multiple sclerosis (MS), suggesting a role of this cytokine in the pathogenesis of the disease. The promoter polymorphism of this gene has been reported to be associated with the severity of atopic and non-atopic asthma in children. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P29460 |
| NCBI GenInfo Identifier: | 266320 |
| NCBI Gene ID: | 3593 |
| NCBI Accession: | P29460.1 |
| UniProt Related Accession: | P29460 |
| Molecular Weight: | 37,169 Da |
| NCBI Full Name: | Interleukin-12 subunit beta |
| NCBI Synonym Full Names: | interleukin 12B |
| NCBI Official Symbol: | IL12BÂ Â |
| NCBI Official Synonym Symbols: | CLMF; NKSF; CLMF2; IMD28; IMD29; NKSF2; IL-12BÂ Â |
| NCBI Protein Information: | interleukin-12 subunit beta; CLMF p40; IL-12 subunit p40; IL12, subunit p40; interleukin 12, p40; interleukin-12 beta chain; NK cell stimulatory factor chain 2; cytotoxic lymphocyte maturation factor 40 kDa subunit; natural killer cell stimulatory factor, 40 kD subunit; interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p40) |
| UniProt Protein Name: | Interleukin-12 subunit beta |
| UniProt Synonym Protein Names: | Cytotoxic lymphocyte maturation factor 40 kDa subunit; CLMF p40; IL-12 subunit p40; NK cell stimulatory factor chain 2; NKSF2 |
| UniProt Gene Name: | IL12BÂ Â |
| UniProt Entry Name: | IL12B_HUMAN |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 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
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| Step | |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
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. |
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