Human Obestatin ELISA Kit

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SKU:
HUFI01732
€599

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Human Obestatin ELISA Kit - Information

The Human Obestatin ELISA Kit can assay for Obestatin in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our 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 Obestatin. During the reaction, Obestatin in the sample or standard competes with a fixed amount of Obestatin on the solid phase supporter for sites on the Biotinylated Detection Antibody specific to 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 Obestatin in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Human Obestatin ELISA Kit - Data

Description
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.
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 (By similarity).
Post-Translational Modification
Uniprot IDQ9UBU3
AliasOB(Obestatin)
Detection methodCompetitiveCompetitive
ApplicationThis immunoassay kit allows for the in vitro quantitative determination of OB concentrations in serum plasma and other biological fluids.
Size96T
Range15.6-1000pg/ml
Sensitivity< 9.375pg/ml
Storage4'C for 6 months
RecoveryMatrices listed below were spiked with certain level of OB and the recovery rates were calculated by comparing the measured value to the expected amount of OB in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)86-10596
EDTA plasma(n=5)85-9891
UFH plasma(n=5)86-9792
LinearityThe linearity of the kit was assayed by testing samples spiked with appropriate concentration of OB and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:81:16
serum(n=5)85-104%88-101%90-104%85-102%
EDTA plasma(n=5)86-98%85-101%87-96%85-100%
UFH plasma(n=5)80-99%81-95%85-100%83-99%
CV(%)Intra-Assay: CV<8%Inter-Assay: CV<10%
NoteFor Research Use Only

Human Obestatin ELISA Kit Protocol

The below protocol is a sample protocol for a Human Obestatin ELISA Kit. Competitive ELISA kits allow for the detection and quantification of an analyte in a sample. This Human Obestatin ELISA Kit allows the researcher to calculate the amount of Human Obestatin 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

ELISA Kit Technical Manual

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.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.

Human Obestatin 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)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:

The ELISA Genie Human Obestatin 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.

Human Obestatin ELISA Kit Protein Information

UniProt Protein Function:leptin: May function as part of a signaling pathway that acts to regulate the size of the body fat depot. An increase in the level of LEP may act directly or indirectly on the CNS to inhibit food intake and/or regulate energy expenditure as part of a homeostatic mechanism to maintain constancy of the adipose mass. Defects in LEP may be a cause of obesity (OBESITY). It is a condition characterized by an increase of body weight beyond the limitation of skeletal and physical requirements, as the result of excessive accumulation of body fat. Belongs to the leptin family.
UniProt Protein Details:

Protein type:Secreted; Secreted, signal peptide; Hormone; Cell development/differentiation

Chromosomal Location of Human Ortholog: 7q31.3

Cellular Component: extracellular space; cytoplasm; extracellular region

Molecular Function:peptide hormone receptor binding; growth factor activity; hormone activity

Biological Process: circadian rhythm; response to dietary excess; positive regulation of myeloid cell differentiation; regulation of fat cell differentiation; regulation of steroid biosynthetic process; female pregnancy; negative regulation of transcription from RNA polymerase II promoter; glucose homeostasis; positive regulation of luteinizing hormone secretion; negative regulation of appetite; positive regulation of tyrosine phosphorylation of Stat3 protein; response to insulin stimulus; response to vitamin E; positive regulation of MAPKKK cascade; regulation of cholesterol absorption; regulation of blood pressure; positive regulation of cell proliferation; positive regulation of ion transport; central nervous system neuron development; placenta development; positive regulation of cytokine production; cholesterol metabolic process; positive regulation of developmental growth; bile acid metabolic process; eating behavior; glucose metabolic process; adult feeding behavior; ovulation from ovarian follicle; leptin-mediated signaling pathway; negative regulation of vasoconstriction; tyrosine phosphorylation of STAT protein; fatty acid beta-oxidation; insulin secretion; glycerol biosynthetic process; energy reserve metabolic process; response to hypoxia; hormone metabolic process; regulation of gluconeogenesis; positive regulation of follicle-stimulating hormone secretion; positive regulation of insulin receptor signaling pathway; leukocyte tethering or rolling; regulation of insulin secretion; negative regulation of apoptosis

Disease: Leptin Deficiency

NCBI Summary:This gene encodes a protein that is secreted by white adipocytes, and which plays a major role in the regulation of body weight. This protein, which acts through the leptin receptor, functions as part of a signaling pathway that can inhibit food intake and/or regulate energy expenditure to maintain constancy of the adipose mass. This protein also has several endocrine functions, and is involved in the regulation of immune and inflammatory responses, hematopoiesis, angiogenesis and wound healing. Mutations in this gene and/or its regulatory regions cause severe obesity, and morbid obesity with hypogonadism. This gene has also been linked to type 2 diabetes mellitus development. [provided by RefSeq, Jul 2008]
UniProt Code:P41159
NCBI GenInfo Identifier:730218
NCBI Gene ID:3952
NCBI Accession:P41159.1
UniProt Secondary Accession:P41159,O15158, Q56A88,
UniProt Related Accession:P41159
Molecular Weight:18,641 Da
NCBI Full Name:Leptin
NCBI Synonym Full Names:leptin
NCBI Official Symbol:LEP Ãƒ‚ 
NCBI Official Synonym Symbols:OB; OBS; LEPD Ãƒ‚ 
NCBI Protein Information:leptin; obese protein; obesity factor; obese, mouse, homolog of; leptin (murine obesity homolog); leptin (obesity homolog, mouse)
UniProt Protein Name:Leptin
UniProt Synonym Protein Names:Obese protein; Obesity factor
Protein Family:Leptin
UniProt Gene Name:LEP Ãƒ‚ 
UniProt Entry Name:LEP_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Competitive
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