Mouse IL-6 ELISA Kit (MOFI00066)
- Product Type:
- ELISA Kit
- 96 Assays
- ELISA Type:
- Sandwich ELISA, Double Antibody
- IL-6, Interleukin 6, IL6, BSF2, HGF, HSF, IFNB2, CDF
|Product Name:||Mouse IL-6 ELISA Kit (MOFI00066)|
|Alias:||IL-6, Interleukin 6, IL6, BSF2, HGF, HSF, IFNB2, CDF|
|Detection Method:||Sandwich ELISA|
|Application:||This immunoassay kit allows for the in vitro quantitative determination of Mouse IL-6 concentrations in serum plasma and other biological fluids.|
|Storage:||4°C for 6 months|
|Note:||For Research Use Only|
|Recovery:||Matrices listed below were spiked with certain level of Mouse IL-6 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse IL-6 in samples.|
|Linearity:||The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse IL-6 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.|
|Intra Assay:||CV <8%|
|Inter Assay:||CV <10%|
|ELISA Microplate (Dismountable)||8×12 strips||4°C for 6 months|
|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)|
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
|UniProt Protein Function:||IL6: Cytokine with a wide variety of biological functions. It is a potent inducer of the acute phase response. Plays an essential role in the final differentiation of B-cells into Ig- secreting cells Involved in lymphocyte and monocyte differentiation. It induces myeloma and plasmacytoma growth and induces nerve cells differentiation Acts on B-cells, T-cells, hepatocytes, hematopoietic progenitor cells and cells of the CNS. Also acts as a myokine. It is discharged into the bloodstream after muscle contraction and acts to increase the breakdown of fats and to improve insulin resistance. Genetic variations in IL6 are associated with susceptibility to rheumatoid arthritis systemic juvenile (RASJ). An inflammatory articular disorder with systemic- onset beginning before the age of 16. It represents a subgroup of juvenile arthritis associated with severe extraarticular features and occasionally fatal complications. During active phases of the disorder, patients display a typical daily spiking fever, an evanescent macular rash, lymphadenopathy, hepatosplenomegaly, serositis, myalgia and arthritis. A IL6 promoter polymorphism is associated with a lifetime risk of development of Kaposi sarcoma in HIV-infected men. Belongs to the IL-6 superfamily.|
|UniProt Protein Details:|
Protein type:Secreted, signal peptide; Secreted
Cellular Component: extracellular space; cell; cytoplasm; extracellular region; interleukin-6 receptor complex; external side of plasma membrane
Molecular Function:protein binding; interleukin-6 receptor binding; growth factor activity; cytokine activity; receptor binding
Biological Process: positive regulation of nitric oxide biosynthetic process; positive regulation of apoptosis; negative regulation of collagen biosynthetic process; positive regulation of transcription, DNA-dependent; response to glucocorticoid stimulus; negative regulation of cytokine secretion; myeloid cell homeostasis; positive regulation of JAK-STAT cascade; glucose homeostasis; positive regulation of tyrosine phosphorylation of Stat3 protein; muscle maintenance; regulation of apoptosis; regulation of cell shape; positive regulation of T-helper 2 cell differentiation; positive regulation of acute inflammatory response; negative regulation of gluconeogenesis; acute-phase response; positive regulation of T cell proliferation; cell growth; defense response to virus; neurite development; positive regulation of protein import into nucleus, translocation; positive regulation of interleukin-6 production; defense response to protozoan; positive regulation of chemokine production; positive regulation of peptidyl-tyrosine phosphorylation; cell redox homeostasis; positive regulation of B cell activation; negative regulation of protein kinase activity; neutrophil apoptosis; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; positive regulation of epithelial cell proliferation; negative regulation of apoptosis; negative regulation of muscle development; T cell activation; positive regulation of translation; negative regulation of hormone secretion; positive regulation of smooth muscle cell proliferation; negative regulation of caspase activity; regulation of circadian sleep/wake cycle, non-REM sleep; negative regulation of cell proliferation; positive regulation of MAPKKK cascade; positive regulation of cell proliferation; response to wounding; hepatic immune response; inflammatory response; negative regulation of chemokine biosynthetic process; cytokine and chemokine mediated signaling pathway; positive regulation of immunoglobulin secretion; positive regulation of peptidyl-serine phosphorylation; endocrine pancreas development; regulation of cell proliferation; positive regulation of protein kinase B signaling cascade; immune response; positive regulation of neuron differentiation; positive regulation of DNA replication; positive regulation of transmission of nerve impulse
|NCBI GenInfo Identifier:||124348|
|NCBI Gene ID:||16193|
|UniProt Secondary Accession:||P08505,Q3UCQ0, Q8BN26,|
|UniProt Related Accession:||P08505|
|Molecular Weight:||24,384 Da|
|NCBI Full Name:||Interleukin-6|
|NCBI Synonym Full Names:||interleukin 6|
|NCBI Official Symbol:||Il6|
|NCBI Official Synonym Symbols:||Il-6|
|NCBI Protein Information:||interleukin-6; interleukin HP-1; B-cell hybridoma growth factor|
|UniProt Protein Name:||Interleukin-6|
|UniProt Synonym Protein Names:||B-cell hybridoma growth factor; Interleukin HP-1|
|UniProt Gene Name:||Il6|
|UniProt Entry Name:||IL6_MOUSE|
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
|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.
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.
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.