Human TRPV1 / Transient receptor potential cation channel subfamily V member 1 ELISA Kit

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SKU:
HUFI01118
€599

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Human TRPV1 / Transient receptor potential cation channel subfamily V member 1 ELISA Kit - Information

The ELISA Genie TRPV1 / Transient receptor potential cation channel subfamily V member 1 ELISA Kit can assay for TRPV1 / Transient receptor potential cation channel subfamily V member 1 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our TRPV1 / Transient receptor potential cation channel subfamily V member 1 ELISA Kits Work?

The ELISA Genie (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, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound TRPV1 / Transient receptor potential cation channel subfamily V member 1 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Human TRPV1 / Transient receptor potential cation channel subfamily V member 1 ELISA Kit - Data

Description

Ligand-activated non-selective calcium permeant cation channel involved in detection of noxious chemical and thermal stimuli. Seems to mediate proton influx and may be involved in intracellular acidosis in nociceptive neurons. Involved in mediation of inflammatory pain and hyperalgesia. Sensitized by a phosphatidylinositol second messenger system activated by receptor tyrosine kinases, which involves PKC isozymes and PCL. Activation by vanilloids, like capsaicin, and temperatures higher than 42 degrees Celsius, exhibits a time- and Ca2+-dependent outward rectification, followed by a long-lasting refractory state. Mild extracellular acidic pH (6.5) potentiates channel activation by noxious heat and vanilloids, whereas acidic conditions (pH <6) directly activate the channel. Can be activated by endogenous compounds, including 12-hydroperoxytetraenoic acid and bradykinin. Acts as ionotropic endocannabinoid receptor with central neuromodulatory effects. Triggers a form of long-term depression (TRPV1-LTD) mediated by the endocannabinoid anandamine in the hippocampus and nucleus accumbens by affecting AMPA receptors endocytosis.

Post-Translational Modification

Phosphorylation by PKA reverses capsaicin-induced dephosphorylation at multiple sites, probably including Ser-117 as a major phosphorylation site. Phosphorylation by CAMKII seems to regulate binding to vanilloids. Phosphorylated and modulated by PRKCE, PRKCM and probably PRKCZ. Dephosphorylation by calcineurin seems to lead to receptor desensitization and phosphorylation by CAMKII recovers activity.

Uniprot ID Q8NER1
Alias

TRPV1/Transient receptor potential cation channel subfamily V member 1/TrpV1/Vanilloid receptor 1/Capsaicin receptor/Osm-9-like TRP channel 1/OTRPC1

Detection method

Sandwich ELISA Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of TRPV1 concentrations in serum plasma and other biological fluids.

Size

96T

Range

0.156-10ng/ml

Sensitivity

< 0.094ng/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of TRPV1 and the recovery rates were calculated by comparing the measured value to the expected amount of TRPV1 in samples.

Matrix Recovery range(%) Average(%)
serum(n=5) 86-100 95
EDTA plasma(n=5) 85-104 95
UFH plasma(n=5) 86-103 95
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of TRPV1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 89-96% 85-93% 88-102% 92-102%
EDTA plasma(n=5) 87-98% 89-97% 90-100% 82-94%
UFH plasma(n=5) 84-98% 89-97% 81-99% 80-93%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Human TRPV1 / Transient receptor potential cation channel subfamily V member 1 ELISA Kit Protocol

The below protocol is a sample protocol for Human TRPV1 / Transient receptor potential cation channel subfamily V member 1 ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human TRPV1 / Transient receptor potential cation channel subfamily V member 1 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.

Sandwich Protocol

Sandwich ELISA Protocol

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.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.

Human TRPV1 / Transient receptor potential cation channel subfamily V member 1 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)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:

The ELISA Genie Human TRPV1 / Transient receptor potential cation channel subfamily V member 1 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 TRPV1 / Transient receptor potential cation channel subfamily V member 1 ELISA Kit Protein Information

UniProt Protein Function:TRPV1: a receptor for capsaicin and a non-selective cation channel that is structurally related to members of the TRP family of ion channels. Activated by increases in temperature in the noxious range, suggesting that it functions as a transducer of painful thermal stimuli in vivo.
UniProt Protein Details:

Protein type:Channel, cation; Membrane protein, multi-pass; Membrane protein, integral; Receptor, misc.

Chromosomal Location of Human Ortholog: 17p13.2

Cellular Component: integral to membrane; integral to plasma membrane; intrinsic to plasma membrane; plasma membrane; postsynaptic membrane

Molecular Function:ATP binding; calcium channel activity; calcium-release channel activity; excitatory extracellular ligand-gated ion channel activity; phosphoprotein binding; transmembrane receptor activity

Biological Process: cell surface receptor linked signal transduction; chemosensory behavior; thermoception; transport

NCBI Summary:Capsaicin, the main pungent ingredient in hot chili peppers, elicits a sensation of burning pain by selectively activating sensory neurons that convey information about noxious stimuli to the central nervous system. The protein encoded by this gene is a receptor for capsaicin and is a non-selective cation channel that is structurally related to members of the TRP family of ion channels. This receptor is also activated by increases in temperature in the noxious range, suggesting that it functions as a transducer of painful thermal stimuli in vivo. Four transcript variants encoding the same protein, but with different 5' UTR sequence, have been described for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:Q8NER1
NCBI GenInfo Identifier:296452849
NCBI Gene ID:7442
NCBI Accession:Q8NER1.2
UniProt Secondary Accession:Q8NER1,Q3LU47, Q9H0G9, Q9H303, Q9H304, Q9NQ74, Q9NY22 A2RUA9,
UniProt Related Accession:Q8NER1
Molecular Weight:94,211 Da
NCBI Full Name:Transient receptor potential cation channel subfamily V member 1
NCBI Synonym Full Names:transient receptor potential cation channel subfamily V member 1
NCBI Official Symbol:TRPV1  
NCBI Official Synonym Symbols:VR1  
NCBI Protein Information:transient receptor potential cation channel subfamily V member 1
UniProt Protein Name:Transient receptor potential cation channel subfamily V member 1
UniProt Synonym Protein Names:Capsaicin receptor; Osm-9-like TRP channel 1; OTRPC1; Vanilloid receptor 1
Protein Family:Transient receptor potential cation channel subfamily
UniProt Gene Name:TRPV1  
UniProt Entry Name:TRPV1_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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