Human GHSR / Growth hormone secretagogue receptor type 1 ELISA Kit

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SKU:
HUFI00411
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q92847
Sensitivity:
0.188ng/ml
Range:
0.313-20ng/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
GHSR, GH-releasing peptide receptor, Ghrelin receptor, GHRP, GHS-R, growth hormone secretagogue receptor, growth hormone secretagogue receptor type 1
Reactivity:
Human
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Description

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Product Name:Human GHSR / Growth hormone secretagogue receptor type 1 ELISA Kit
Product Code:HUFI00411
Size:96 Assays
Alias:GHSR, GH-releasing peptide receptor, Ghrelin receptor, GHRP, GHS-R, growth hormone secretagogue receptor, growth hormone secretagogue receptor type 1
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human GHSR concentrations in serum plasma and other biological fluids.
Sensitivity:0.188ng/ml
Range:0.313-20ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human GHSR and the recovery rates were calculated by comparing the measured value to the expected amount of Human GHSR in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)88-10296
EDTA plasma(n=5)86-10394
UFH plasma(n=5)91-9994
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human GHSR and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)95-103%98-103%87-105%
EDTA plasma(n=5)84-98%93-101%85-101%
UFH plasma(n=5)84-98%82-98%81-97%
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)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:

  • 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
UniprotQ92847
UniProt Protein Function:GHSR: Receptor for ghrelin, coupled to G-alpha-11 proteins. Stimulates growth hormone secretion. Binds also other growth hormone releasing peptides (GHRP) (e.g. Met-enkephalin and GHRP-6) as well as non-peptide, low molecular weight secretagogues (e.g. L-692,429, MK-0677, adenosine). Defects in GHSR may be a cause of idiopathic short stature autosomal (ISSA). Short stature is defined by a subnormal rate of growth. Belongs to the G-protein coupled receptor 1 family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:Protein type:Receptor, GPCR; Membrane protein, integral; Membrane protein, multi-pass; GPCR, family 1

Chromosomal Location of Human Ortholog: 3q26.31

Cellular Component: neuron projection; cell surface; plasma membrane; integral to membrane; lipid raft

Molecular Function:G-protein coupled receptor activity; growth hormone secretagogue receptor activity; peptide hormone binding; growth hormone-releasing hormone receptor activity

Biological Process: response to food; positive regulation of insulin-like growth factor receptor signaling pathway; hormone-mediated signaling; response to hormone stimulus; negative regulation of interleukin-6 biosynthetic process; adult feeding behavior; positive regulation of multicellular organism growth; decidualization; negative regulation of interleukin-1 beta production; negative regulation of insulin secretion; growth hormone secretion; G-protein coupled receptor protein signaling pathway; positive regulation of fatty acid metabolic process; regulation of synaptogenesis; cellular response to insulin stimulus; negative regulation of inflammatory response; actin polymerization and/or depolymerization; positive regulation of appetite; regulation of hindgut contraction; negative regulation of tumor necrosis factor biosynthetic process

Disease: Short Stature, Idiopathic, Autosomal

NCBI Summary:This gene encodes a member of the G-protein coupled receptor family. The encoded protein may play a role in energy homeostasis and regulation of body weight. Two identified transcript variants are expressed in several tissues and are evolutionary conserved in fish and swine. One transcript, 1a, excises an intron and encodes the functional protein; this protein is the receptor for the Ghrelin ligand and defines a neuroendocrine pathway for growth hormone release. The second transcript (1b) retains the intron and does not function as a receptor for Ghrelin; however, it may function to attenuate activity of isoform 1a. Mutations in this gene are associated with autosomal idiopathic short stature.[provided by RefSeq, Apr 2010]
UniProt Code:Q92847
NCBI GenInfo Identifier:2494998
NCBI Gene ID:2693
NCBI Accession:Q92847.1
UniProt Secondary Accession:Q92847,Q14D12, Q6ISR8, Q92848, Q96RJ7,
UniProt Related Accession:Q92847
Molecular Weight:289
NCBI Full Name:Growth hormone secretagogue receptor type 1
NCBI Synonym Full Names:growth hormone secretagogue receptor
NCBI Official Symbol:GHSR  
NCBI Protein Information:growth hormone secretagogue receptor type 1; GHRP; GHS-R; ghrelin receptor; GH-releasing peptide receptor
UniProt Protein Name:Growth hormone secretagogue receptor type 1
UniProt Synonym Protein Names:GH-releasing peptide receptor; GHRP; Ghrelin receptor
Protein Family:Growth hormone secretagogue receptor
UniProt Gene Name:GHSR  
UniProt Entry Name:GHSR_HUMAN

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

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.

StepProtocol
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.

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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