Human TACE / ADAM17 ELISA Kit

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SKU:
HUFI01209
€599

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Human TACE / ADAM17 ELISA Kit - Information

The ELISA Genie TACE / ADAM17 ELISA Kit can assay for TACE / ADAM17 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our TACE / ADAM17 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 TACE / ADAM17 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 TACE / ADAM17 ELISA Kit - Data

Description

Cleaves the membrane-bound precursor of TNF-alpha to its mature soluble form. Responsible for the proteolytical release of soluble JAM3 from endothelial cells surface. Responsible for the proteolytic release of several other cell-surface proteins, including p75 TNF-receptor, interleukin 1 receptor type II, p55 TNF-receptor, transforming growth factor-alpha, L-selectin, growth hormone receptor, MUC1 and the amyloid precursor protein. Acts as an activator of Notch pathway by mediating cleavage of Notch, generating the membrane-associated intermediate fragment called Notch extracellular truncation (NEXT). Plays a role in the proteolytic processing of ACE2.

Post-Translational Modification

The precursor is cleaved by a furin endopeptidase. Phosphorylated. Stimulation by growth factor or phorbol 12-myristate 13-acetate induces phosphorylation of Ser-819 but decreases phosphorylation of Ser-791. Phosphorylation at THR-735 by MAPK14 is required for ADAM17-mediated ectodomain shedding.

Uniprot ID P78536
Alias

TACE(TNF alpha Converting Enzyme)/ADAM17/CD156b/CSVP/ADAM18/CD156b antigen/ADAM metallopeptidase domain 18/disintegrin and metalloproteinase domain-containing protein 17/Snake venom-like protease/TNF-alpha convertase/TNF-alpha converting enzyme/TNF-alpha-converting enzyme

Detection method

Sandwich ELISA Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of ADAM17 concentrations in serum plasma and other biological fluids.

Size

96T

Range

62.5-4000pg/ml

Sensitivity

< 37.5pg/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of ADAM17 and the recovery rates were calculated by comparing the measured value to the expected amount of ADAM17 in samples.

Matrix Recovery range(%) Average(%)
serum(n=5) 86-105 93
EDTA plasma(n=5) 89-103 94
UFH plasma(n=5) 88-99 91
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of ADAM17 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) 92-102% 86-101% 85-104% 86-104%
EDTA plasma(n=5) 82-94% 83-101% 85-99% 83-100%
UFH plasma(n=5) 80-89% 81-84% 87-98% 83-99%
CV(%)

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

Note

For Research Use Only

Human TACE / ADAM17 ELISA Kit Protocol

The below protocol is a sample protocol for Human TACE / ADAM17 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 TACE / ADAM17 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 TACE / ADAM17 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 TACE / ADAM17 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 TACE / ADAM17 ELISA Kit Protein Information

UniProt Protein Function:TACE: a type I membrane protein with disintegrin and metalloprotease activity. An ubiquitously expressed ectoenzyme of peptidase family M12B. Must be membrane anchored to cleave the different substrates. The cytoplasmic domain is not required for the this activity. Possesses a narrow endopeptidase specificity. Cleaves Pro-Leu-Ala-Gln-Ala-|-Val-Arg-Ser-Ser-Ser in the membrane-bound, 26-kDa form of tumor necrosis factor alpha (TNF-alpha) to its mature soluble form. Responsible for the proteolytic release of several other cell-surface proteins, including p75 TNF-receptor, interleukin 1 receptor type II, p55 TNF-receptor, transforming growth factor-alpha, L-selectin, and the amyloid precursor protein. Also involved in the activation of Notch pathway. Binds 1 zinc ion per subunit. Two splice variant isoforms have been described.
UniProt Protein Details:

Protein type:Motility/polarity/chemotaxis; EC 3.4.24.86; Protease; Membrane protein, integral

Chromosomal Location of Human Ortholog: 2p25

Cellular Component: cell surface; focal adhesion; membrane; integral to plasma membrane; apical plasma membrane; cytoplasm; plasma membrane; intercellular junction; actin cytoskeleton; lipid raft

Molecular Function:integrin binding; protein binding; interleukin-6 receptor binding; zinc ion binding; metallopeptidase activity; metalloendopeptidase activity; Notch binding; SH3 domain binding; PDZ domain binding

Biological Process: extracellular matrix organization and biogenesis; nerve growth factor receptor signaling pathway; cell motility involved in cell locomotion; neutrophil mediated immunity; T cell differentiation in the thymus; response to lipopolysaccharide; positive regulation of leukocyte chemotaxis; proteolysis; G1/S-specific positive regulation of cyclin-dependent protein kinase activity; extracellular matrix disassembly; positive regulation of epidermal growth factor receptor activity; positive regulation of cell proliferation; negative regulation of interleukin-8 production; germinal center formation; cell adhesion; response to drug; spleen development; epidermal growth factor receptor signaling pathway; Notch signaling pathway; membrane protein intracellular domain proteolysis; response to high density lipoprotein stimulus; membrane protein ectodomain proteolysis; positive regulation of transforming growth factor beta receptor signaling pathway; Notch receptor processing; positive regulation of cell motility; positive regulation of cell growth; positive regulation of chemokine production; collagen catabolic process; regulation of mast cell apoptosis; PMA-inducible membrane protein ectodomain proteolysis; B cell differentiation; response to hypoxia; cell adhesion mediated by integrin; positive regulation of protein amino acid phosphorylation; negative regulation of transforming growth factor beta receptor signaling pathway; wound healing, spreading of epidermal cells; positive regulation of cell migration

Disease: Inflammatory Skin And Bowel Disease, Neonatal, 1

NCBI Summary:This gene encodes a member of the ADAM (a disintegrin and metalloprotease domain) family. Members of this family are membrane-anchored proteins structurally related to snake venom disintegrins, and have been implicated in a variety of biologic processes involving cell-cell and cell-matrix interactions, including fertilization, muscle development, and neurogenesis. The protein encoded by this gene functions as a tumor necrosis factor-alpha converting enzyme; binds mitotic arrest deficient 2 protein; and also plays a prominent role in the activation of the Notch signaling pathway. [provided by RefSeq, Jul 2008]
UniProt Code:P78536
NCBI GenInfo Identifier:73747889
NCBI Gene ID:6868
NCBI Accession:NP_003174.3
UniProt Secondary Accession:P78536,O60226,
UniProt Related Accession:P78536
Molecular Weight:93,021 Da
NCBI Full Name:disintegrin and metalloproteinase domain-containing protein 17 preproprotein
NCBI Synonym Full Names:ADAM metallopeptidase domain 17
NCBI Official Symbol:ADAM17  
NCBI Official Synonym Symbols:CSVP; TACE; NISBD; ADAM18; CD156B  
NCBI Protein Information:disintegrin and metalloproteinase domain-containing protein 17; TNF-alpha convertase; snake venom-like protease; TNF-alpha converting enzyme; ADAM metallopeptidase domain 18; tumor necrosis factor, alpha, converting enzyme
UniProt Protein Name:Disintegrin and metalloproteinase domain-containing protein 17
UniProt Synonym Protein Names:Snake venom-like protease; TNF-alpha convertase; TNF-alpha-converting enzyme
Protein Family:Disintegrin and metalloproteinase domain-containing protein
UniProt Gene Name:ADAM17  
UniProt Entry Name:ADA17_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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