Rat IL-1B ELISA Kit

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SKU:
RTFI00904
€299

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Rat IL 1B ELISA Kit - Information

The ELISA Genie Rat IL1B / IL1 beta ELISA Kit can assay for Rat IL1B / IL1 beta in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our Rat IL1B / IL1 beta ELISA Kits Work?

The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound Rat IL1B / IL1 beta is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Rat IL 1B ELISA Kit - Data

Description

Potent proinflammatory cytokine. Initially discovered as the major endogenous pyrogen, induces prostaglandin synthesis, neutrophil influx and activation, T-cell activation and cytokine production, B-cell activation and antibody production, and fibroblast proliferation and collagen production.

Post-Translational Modification

Uniprot ID Q63264
Detection method

Sandwich ELISA Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of IL-1 beta concentrations in serum plasma and other biological fluids.

Size

96T

Range

31.25-2000pg/ml

Sensitivity

< 18.75pg/ml

Storage

4'C for 6 months

Recovery

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

Matrix Recovery range(%) Average(%)
serum(n=5) 87-105 95
EDTA plasma(n=5) 87-99 93
UFH plasma(n=5) 86-104 95
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of IL-1 beta and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 85-101% 86-96% 85-105% 88-105%
EDTA plasma(n=5) 88-98% 83-99% 85-100% 86-101%
UFH plasma(n=5) 83-92% 84-99% 84-95% 87-97%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Rat IL1B ELISA Kit Protocol

The below protocol is a sample protocol for Rat IL1B ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Rat IL1B present in their sample.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 ‚°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Sandwich Protocol

Sandwich ELISA Protocol

Kit Protocol:

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.

Rat IL1B ELISA Kit components

96 Assays

Storage

ELISA Microplate(Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The ELISA Genie Rat IL1B ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • 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

Sample Preparation

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 TypeProtocol

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.

Rat IL1B ELISA Kit Protein Information

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; extracellular region; vesicle; secretory granule

Molecular Function:protein domain specific binding; interleukin-1 receptor binding; cytokine activity

Biological Process: positive regulation of granulocyte macrophage colony-stimulating factor production; negative regulation of MAP kinase activity; positive regulation of JNK activity; positive regulation of nitric oxide biosynthetic process; negative regulation of glutamate secretion; glycoprotein metabolic process; positive regulation of apoptosis; activation of MAPK activity; positive regulation of transcription, DNA-dependent; positive regulation of interleukin-2 biosynthetic process; response to glucocorticoid stimulus; germ cell programmed cell death; negative regulation of insulin receptor signaling pathway; positive regulation of glial cell differentiation; positive regulation of NF-kappaB import into nucleus; response to lipopolysaccharide; positive regulation of lipid catabolic process; fever; positive regulation of membrane protein ectodomain proteolysis; response to organic cyclic substance; response to carbohydrate stimulus; activation of NF-kappaB transcription factor; elevation of cytosolic calcium ion concentration; response to vitamin D; pentacyclic triterpenoid metabolic process; positive regulation of phagocytosis; positive regulation of T cell proliferation; positive regulation of astrocyte differentiation; response to drug; 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; response to organic nitrogen; purine base metabolic process; positive regulation of angiogenesis; negative regulation of neuron differentiation; response to ethanol; response to heat; 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; response to peptide hormone stimulus; estrogen metabolic process; sequestering of triacylglycerol; wound healing; positive regulation of interleukin-6 biosynthetic process; response to morphine; positive regulation of JNK cascade; negative regulation of transcription from RNA polymerase II promoter; response to L-ascorbic acid; chronic inflammatory response to antigenic stimulus; positive regulation of stress-activated MAPK cascade; response to estradiol stimulus; negative regulation of neurogenesis; positive regulation of interleukin-8 production; negative regulation of cell proliferation; learning and/or memory; negative regulation of lipid catabolic process; hyaluronan biosynthetic process; protein kinase B signaling cascade; lipopolysaccharide-mediated signaling pathway; response to gamma radiation; regulation of I-kappaB kinase/NF-kappaB cascade; inflammatory response; response to nutrient; aging; cytokine and chemokine mediated signaling pathway; MAPKKK cascade; positive regulation of immature T cell proliferation in the thymus; response to ATP; memory; ovulation; polyketide metabolic process; positive regulation of interferon-gamma production; positive regulation of chemokine biosynthetic process; response to ozone; positive regulation of prostaglandin secretion; response to hypoxia; positive regulation of fever; immune response; positive regulation of protein amino acid phosphorylation; regulation of insulin secretion

NCBI Summary:an inflammatory cytokine [RGD, Feb 2006]
UniProt Code:Q63264
NCBI GenInfo Identifier:2497335
NCBI Gene ID:24494
NCBI Accession:Q63264.1
UniProt Related Accession:Q63264
Molecular Weight:30,644 Da
NCBI Full Name:Interleukin-1 beta
NCBI Synonym Full Names:interleukin 1 beta
NCBI Official Symbol:Il1b  
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_RAT
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Additional Information

Product type:
ELISA
Reactivity:
Rat
ELISA Type:
Sandwich
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