Description
Rat Ghrelin ELISA Kit
The Rat Ghrelin ELISA Kit is specifically designed for the accurate and sensitive detection of ghrelin levels in rat serum, plasma, and tissue culture supernatants. Ghrelin is a peptide hormone that plays a key role in regulating appetite, energy metabolism, and growth hormone secretion.This ELISA kit offers high sensitivity and specificity, ensuring precise and reproducible results for a wide range of research applications. Ghrelin has been implicated in various physiological processes, including food intake, glucose homeostasis, and gastrointestinal motility, making it a valuable biomarker for studying metabolic disorders, obesity, and other related conditions.
With this kit, researchers can easily measure ghrelin levels in rat samples, helping to advance our understanding of ghrelin's role in health and disease and potentially leading to the development of innovative therapies targeting ghrelin signaling pathways.
| Product Name: | Rat Ghrelin ELISA Kit |
| Product Code: | RTFI00478 |
| Size: | 96 Assays |
| Target: | Rat Ghrl |
| Alias: | Ghrl, ghrelin, ghrelin, obestatin preprohormone, ghrelin, obestatin prepropeptide, Growth hormone secretagogue, Growth hormone-releasing peptide, Motilin-related peptide, MTLRPgrowth hormone secretagogue receptor ligand, obestatin, Protein M46 |
| Reactivity: | Rat |
| Detection Method: | Competitive ELISA, Coated with Antibody |
| Sensitivity: | 2.813pg/ml |
| Range: | 4.688-300pg/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Rat Ghrl and the recovery rates were calculated by comparing the measured value to the expected amount of Rat Ghrl in samples. | ||||||||||||||||
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat Ghrl and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. | ||||||||||||||||
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| Intra-Assay: | CV <8% | ||||||||||||||||
| Inter-Assay: | CV <10% |
| Uniprot: | Q9QYH7 |
| 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] |
| UniProt Code: | Q9QYH7 |
| NCBI GenInfo Identifier: | 11067387 |
| NCBI Gene ID: | 59301 |
| NCBI Accession: | NP_067701.1 |
| 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 |
| 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. | Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank or Sample per well. The blank well is added with Sample Dilution Buffer. Immediately add 50 µL of biotin-labelled antibody working solution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thorough mixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA plate well, avoid touching plate walls and foaming). |
| 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 30 minutes 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 30 minutes. 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. |
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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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. |
