Mitogen Activited Protein Kinase (MAPK) ELISA Kit

Antigen: Mitogen Activited Protein Kinase (MAPK)

Reaktivität: Human

Applikation: ELISA

Zertifikate: ISO 9001:2008

Produktnummer: ABIN366960

Produktbeschreibung

Produktmerkmale: This immunoassay kit allows for the in vitro quantitative determination of human MAPK concentrations in cell culture supernates, serum, plasma and other biological fluids.

Proben: Serum, Plasma

Beschreibung: Mitogen-activated protein (MAP) kinases (EC 2.7.11.24) are serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens) and regulate various cellular activities, such as gene expression, mitosis, differentiation, proliferation, and cell survival/apoptosis. MAPKs are involved in the action of most nonnuclear oncogenes. They are involved in cell response to growth factors such as BDNF or nerve growth factor. Extracellular stimuli lead to activation of a MAP kinase via a signaling cascade ("MAPK cascade") composed of MAP kinase, MAP kinase kinase (MKK, MEKK, or MAP2K), and MAP kinase kinase kinase (MKKK or MAP3K, EC 2.7.11.25). A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues, and then this MAP2K activates a MAP kinase through phosphorylation on its serine and tyrosine residues. (ie MAP2K can be a dual-specificity kinase). This MAP kinase signaling cascade has been evolutionarily well-conserved from yeast to mammals.

Spezifität: This assay recognizes recombinant and natural human MAPK. No significant cross-reactivity or interference was observed.

Anwendungen

Prinzip: The microtiter plate provided in this kit has been pre-coated with an antibody specific to human MAPK. Standards or samples are then added to the appropriate microtiter plate wells with a HRP-conjugated antibody preparation specific for human MAPK and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of human MAPK in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protokoll: Assay Procedure:
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate . 1. Set a Blank well without any solution. Add 50μl of Standard or Sample per well. Standard need test in duplicate. Incubate for 30mins at 37° C. 2. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (200μl) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. 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 blot it against clean paper towels. 7 3. Add 50μl of HRP-conjugate to each well (not to Blank well). Mix well and then incubate for 45mins at 37°C. 4. Fill each well with Wash Buffer (about 200μl), stay for 10 seconds and Spinning. Repeat the process for a total of three washes. 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 blot it against clean paper towels. 5. Add 50μl of Substrate A and Substrate B to each well, mix well. Incubate for 15 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 6. Add 50μl of Stop Solution to each well. 7. Determine the optical density of each well within 10 minutes, using a microplate reader set to 450 nm.
Calculation of Results:
Average the duplicate readings for each standard, Blank, and sample and subtract the optical density of Blank. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the human MAPK concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. 8.
Limitations of the Procedure:
The kit should not be used beyond the expiration date on the kit label.Do not mix or substitute reagents with those from other lots or sources.It is important that the Calibrator Diluent selected for the standard curve be consistent with the samples being assayed.If samples generate values higher than the highest standard, dilute the samples with the appropriate Calibrator Diluent and repeat the assay.Any variation in Standard Diluent, operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding.This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
Sample collection and storage:
Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Note: Grossly hemolyzed samples are not suitable for use in this assay.

Testdurchführung: Assay Time: 1-3h
Sample Volume: 50-100ul

Applikationshinweise: Detection Wavelength: 450 nm

Bestandteile: Reagent (Quantity): Assay plate (1), Standard (5), HRP-conjugate (1x6ml), Wash Buffer (20×concentrate) (1x15ml), Substrate A (1x7ml), Substrate B (1x7ml), Stop Solution (1x7ml)

Benötigtes Material: Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. Pipettes and pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer.

Lagerung: 1. Unopened test kits should be stored at 2-8 C upon receipt and the microtiter plate should be kept in a sealed bag to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement. 5

Beschränkungen: Nur für Forschungszwecke einsetzbar