Human Ghrelin ELISA Kit

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SKU:
HUFI00296
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9UBU3
Sensitivity:
1.125pg/ml
Range:
1.875-120pg/ml
ELISA Type:
Competitive ELISA, Coated with Antigen
Synonyms:
GHRL, Ghrelin, Obestatin
Reactivity:
Human
Frequently bought together:

Description

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Product Name:Human Ghrelin ELISA Kit
Product Code:HUFI00296
Size:96 Assays
Alias:GHRL, Ghrelin, Obestatin
Detection method:Competitive ELISA, Coated with Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human GHRL concentrations in serum plasma and other biological fluids.
Sensitivity:1.125pg/ml
Range:1.875-120pg/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human GHRL and the recovery rates were calculated by comparing the measured value to the expected amount of Human GHRL in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)89-9994
EDTA plasma(n=5)90-10496
UFH plasma(n=5)86-10296
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human GHRL and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-96%88-97%87-101%
EDTA plasma(n=5)87-101%83-100%82-98%
UFH plasma(n=5)83-100%86-98%85-96%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate(Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)60ul4°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:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipettetips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir
UniprotQ9UBU3
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; Secreted, signal peptide; Cell development/differentiation; Cell cycle regulation; Apoptosis

Chromosomal Location of Human Ortholog: 3p26-p25

Cellular Component: extracellular space; axon; endoplasmic reticulum lumen; extracellular region

Molecular Function:growth hormone-releasing hormone activity; G-protein-coupled receptor binding; ghrelin receptor binding; protein tyrosine kinase activator activity

Biological Process: cortisol secretion; negative regulation of circadian sleep/wake cycle, REM sleep; positive regulation of cortisol secretion; hormone-mediated signaling; activation of MAPK activity; response to hormone stimulus; negative regulation of interleukin-6 biosynthetic process; decidualization; positive regulation of multicellular organism growth; positive regulation of adrenocorticotropic hormone secretion; negative regulation of insulin secretion; growth hormone secretion; elevation of cytosolic calcium ion concentration; negative regulation of locomotion; positive regulation of appetite; dendrite development; negative regulation of tumor necrosis factor biosynthetic process; positive regulation of circadian sleep/wake cycle, non-REM sleep; regulation of response to food; gastric acid secretion; positive regulation of insulin secretion; glucose metabolic process; adult feeding behavior; negative regulation of interleukin-1 beta production; regulation of cell proliferation; positive regulation of growth hormone secretion; positive regulation of synaptogenesis; G-protein coupled receptor protein signaling pathway; negative regulation of angiogenesis; cellular protein metabolic process; response to estrogen stimulus; negative regulation of inflammatory response; actin polymerization and/or depolymerization; cartilage development; negative regulation of endothelial cell proliferation; regulation of excitatory postsynaptic membrane potential; negative regulation of apoptosis

Disease: Obesity

NCBI Summary:This gene encodes ghrelin-obestatin preproprotein, which generates ghrelin and obestatin. Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor and is involved in regulating growth hormone release. Obestatin was initially reported to be an endogenous ligand for the orphan G protein-coupled receptor GPR39 and was involved in satiety and decreased food intake; however, these findings are controversial. Recent reports show that obestatin is involved in inhibiting thirst and anxiety, improving memory, regulating sleep, affecting cell proliferation, and increasing the secretion of pancreatic juice enzymes. Alternative promoters and alternative splicing result in multiple transcript variants, some of which encode different protein isoforms and some of which do not encode a protein but may regulate the ghrelin-obestatin preproprotein expression. In addition, antisense transcripts for this gene have been identified and may also function in regulation of the ghrelin-obestatin preproprotein expression. [provided by RefSeq, Oct 2008]
UniProt Code:Q9UBU3
NCBI GenInfo Identifier:17865471
NCBI Gene ID:51738
NCBI Accession:Q9UBU3.1
UniProt Secondary Accession:Q9UBU3,Q86YP8, Q8TAT9, Q9H3R3, A8CF34, A8CF38, A8CF42 A8DN29, A8DN30,
UniProt Related Accession:Q9UBU3
Molecular Weight:9,972 Da
NCBI Full Name:Appetite-regulating hormone
NCBI Synonym Full Names:ghrelin/obestatin prepropeptide
NCBI Official Symbol:GHRL  
NCBI Official Synonym Symbols:MTLRP  
NCBI Protein Information:appetite-regulating hormone; motilin-related peptide; growth hormone secretagogue; ghrelin/obestatin preprohormone; growth hormone-releasing peptide; prepro-appetite regulatory hormone; ghrelin, growth hormone secretagogue receptor ligand
UniProt Protein Name:Appetite-regulating hormone
UniProt Synonym Protein Names:Growth hormone secretagogue; Growth hormone-releasing peptide; Motilin-related peptide; Protein M46Cleaved into the following 3 chains:Ghrelin-27; Ghrelin-28; Ghrelin; Obestatin
Protein Family:Ancylostoma secreted protein
UniProt Gene Name:GHRL  
UniProt Entry Name:GHRL_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Equilibrate the TMB substrate for at least 30 min at 37°C beforeuse. When diluting samples and reagents, they must be mixed completely andevenly. It is recommended to plot a standard curve for each test.

StepProtocol
1.Set standard, test sample and control (zero) wells on the pre-coatedplate respectively, and then, record their positions. It isrecommended to measure each standard and sample in duplicate. Washplate 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 blankwell is added with Sample Dilution Buffer. Immediately add 50 µL of biotin-labelled antibody workingsolution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thoroughmixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA platewell, avoid touching plate walls and foaming).
3.Wash: Aspirate each well and wash, repeating the process three timesWash by filling each well with Wash Buffer (approximately 350µL)using a squirt bottle, multi-channel pipette, manifold dispenser orautomated washer. Complete removal of liquid at each step is essentialto good performance. After the last wash, remove any remaining WashBuffer by aspirating or decanting. Invert the plate and pat it againstthick clean absorbent paper.
4.HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC workingsolution to each well. Cover with a new Plate sealer. Incubate for30minutes at 37°C.
5.Wash: Repeat the aspiration/wash process for five times.
6.TMB Substrate: Add 90µL of TMB Substrate to each well. Coverwith a new Plate sealer. Incubate for about 10-20 minutes at 37°C.Protect from light. The reaction time can be shortened or extendedaccording to the actual color change, but not more than 30minutes.When apparent gradient appeared in standard wells, you can terminatethe reaction.
7.Stop: Add 50µL of Stop Solution to each well. Color turn toyellow immediately. The adding order of stop solution should be as thesame as the substrate solution.
8.OD Measurement: Determine the optical density (OD Value) of each wellat once, using a microplate reader set to 450 nm. You should open themicroplate 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 TypeProtocol
SerumIf 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.

PlasmaCollect 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 FluidCollect 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 supernatantCollect 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 lysatesSolubilize 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 homogenatesThe 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 lysatesRinse 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 MilkCollect 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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