Rat Ghrelin ELISA Kit
Rat Ghrelin ELISA Kit - Information
The Rat Ghrelin ELISA Kit can assay for Rat Ghrelin / Obestatin in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.How our Rat Ghrelin / Obestatin ELISA Kits Work?
The ELISA Genie ELISA (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, We have developed a range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands.
This ELISA kit uses Competitive-ELISA as the method. The microtiter plate provided in this kit has been pre-coated with Rat Ghrelin / Obestatin. During the reaction, Rat Ghrelin / Obestatin in the sample or standard competes with a fixed amount of Rat Ghrelin / Obestatin on the solid phase supporter for sites on the Biotinylated Detection Antibody specific to Rat Ghrelin / Obestatin. Excess conjugate and unbound sample or standard are washed from the plate, and HRP-Streptavidin(SABC) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Rat Ghrelin / Obestatin in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Rat Ghrelin ELISA Kit - Data
Ghrelin is the ligand for growth hormone secretagogue receptor type 1 (GHSR). Induces the release of growth hormone from the pituitary. Has an appetite-stimulating effect, induces adiposity and stimulates gastric acid secretion. Involved in growth regulation.;_x000D_ Obestatin may be the ligand for GPR39. May have an appetite-reducing effect resulting in decreased food intake. May reduce gastric emptying activity and jejunal motility.
|Post-Translational Modification|| |
O-octanoylation is essential for ghrelin activity. The replacement of Ser-26 by aromatic tryptophan preserves ghrelin activity (PubMed:10604470, PubMed:10801861)._x000D_ Amidation of Leu-98 is essential for obestatin activity.
|Detection method|| |
This immunoassay kit allows for the in vitro quantitative determination of Ghrl concentrations in serum plasma and other biological fluids.
4'C for 6 months
Matrices listed below were spiked with certain level of Ghrl and the recovery rates were calculated by comparing the measured value to the expected amount of Ghrl in samples.
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Ghrl and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
For Research Use Only
Rat Ghrelin ELISA Kit Protocol
The below protocol is a sample protocol for a Rat Ghrelin ELISA Kit. Competitive ELISA kits allow for the detection and quantification of an analyte in a sample. This Rat Ghrelin ELISA Kit allows the researcher to calculate the amount of Rat Ghrelin 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.
Competitive ELISA 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.||Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank, or Sample per well. The blank well is added with Sample / Standard dilution buffer. Immediately add 50 µL of Biotin-detection antibody working solution to each well. Cover with the Plate sealer we provided. Gently tap the plate to ensure thorough mixing. Incubate for 45minutes at 37°C. (Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming to the best of your ability.)|
|3.||Wash: Aspirate each well and wash, repeating the process three times Wash by filling each well with Wash Buffer (approximately 350µL) using a squirt bottle, multi-channel pipette, manifold dispenser or automated washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.|
|4.||HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC working solution to each well. Cover with a new Plate sealer. Incubate for 30minutes at 37°C.|
|5.||Wash: Repeat the aspiration/wash process for five times.|
|6.||TMB Substrate: Add 90µL of TMB Substrate to each well. Cover with a new Plate sealer. Incubate for about 10-20 minutes at 37°C. Protect from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30minutes. When apparent gradient appeared in standard wells, you can terminate the reaction.|
|7.||Stop: Add 50µL of Stop Solution to each well. Color turn to yellow immediately. The adding order of stop solution should be as the same as the substrate solution.|
|8.||OD Measurement: Determine the optical density (OD Value) of each well at once, using a microplate reader set to 450 nm. You should open the microplate reader ahead, preheat the instrument, and set the testing parameters.|
Rat Ghrelin ELISA Kit components
|ELISA Microplate(Dismountable)||8×12 strips||4°C for 6 months|
|Sample/Standard Dilution Buffer||20ml||4°C|
|Biotin-labeled Antibody(Concentrated)||60ul||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:The ELISA Genie Rat Ghrelin 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
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
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.
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.
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.
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.
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 Ghrelin ELISA Kit Protein Information
|UniProt Protein Function:||ghrelin: a hormone that binds to the growth hormone secretagogue receptor type 1 (GHSR). Secreted by the stomach|
|UniProt Protein Details:|
Protein type:Secreted, signal peptide; Cell development/differentiation; Secreted; Cell cycle regulation; Apoptosis
Cellular Component: axon; cytoplasm; cytosol; extracellular region; extracellular space
Molecular Function:G-protein-coupled receptor binding; ghrelin receptor binding; growth hormone-releasing hormone activity; hormone activity; protein tyrosine kinase activator activity
Biological Process: actin polymerization and/or depolymerization; activation of MAPK activity; adult feeding behavior; decidualization; dendrite development; elevation of cytosolic calcium ion concentration; G-protein coupled receptor protein signaling pathway; gastric acid secretion; hormone-mediated signaling; negative regulation of apoptosis; negative regulation of circadian sleep/wake cycle, REM sleep; negative regulation of endothelial cell proliferation; negative regulation of inflammatory response; negative regulation of insulin secretion; negative regulation of interleukin-1 beta production; negative regulation of interleukin-6 biosynthetic process; negative regulation of locomotion; negative regulation of tumor necrosis factor biosynthetic process; positive regulation of adrenocorticotropic hormone secretion; positive regulation of appetite; positive regulation of circadian sleep/wake cycle, non-REM sleep; positive regulation of cortisol secretion; positive regulation of growth hormone secretion; positive regulation of insulin secretion; positive regulation of response to food; positive regulation of synaptogenesis; regulation of cell proliferation; regulation of excitatory postsynaptic membrane potential; regulation of response to food; response to estrogen stimulus; response to hormone stimulus
|NCBI Summary:||endogenous ligand for the growth hormone secretagogue receptor (GHSR); involved in testicular function and GH release [RGD, Feb 2006]|
|NCBI GenInfo Identifier:||11067387|
|NCBI Gene ID:||59301|
|UniProt Secondary Accession:||Q9QYH7,Q9ET69,|
|UniProt Related Accession:||Q9QYH7|
|Molecular Weight:||13,048 Da|
|NCBI Full Name:||appetite-regulating hormone preproprotein|
|NCBI Synonym Full Names:||ghrelin/obestatin prepropeptide|
|NCBI Official Symbol:||Ghrl|
|NCBI Protein Information:||appetite-regulating hormone|
|UniProt Protein Name:||Appetite-regulating hormone|
|UniProt Synonym Protein Names:||Growth hormone secretagogue; Growth hormone-releasing peptide; Motilin-related peptide|
|Protein Family:||Appetite-regulating hormone|
|UniProt Gene Name:||Ghrl|
|UniProt Entry Name:||GHRL_RAT|