We are preparing the requested document. Please wait, this may take a while...!
|Antigen||Protein Phosphatase 3, Regulatory Subunit B, alpha (PPP3R1) ELISA Kits|
|Reaktivität||Ratte (Rattus) Alternativen|
Kits mit alternativen Reaktivitäten:
|Untere Nachweisgrenze||0.156 ng/mL|
|Hersteller||Anmelden zum Anzeigen|
Produktdetails PPP3R1 ELISA KitAntigendetails Anwendungsinformationen Handhabung Bilder
|Verwendungszweck||This immunoassay kit allows for the specific measurement of rat calcineurin, CaN concentrations in cell culture supernates, serum, plasma and other relevant liquid.|
|Proben||Cell Culture Supernatant, Serum, Plasma, Biological Fluids|
|Spezifität||This assay recognizes recombinant and natural rat CaN.|
|Kreuzreaktivität (Details)||No significant cross-reactivity or interference was observed.|
|Sensitivität||The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.|
|Produktmerkmale||Rattus norvegicus,Rat,Calcineurin subunit B type 1,Protein phosphatase 2B regulatory subunit 1,Protein phosphatase 3 regulatory subunit B alpha isoform 1,Ppp3r1,Cna2,Cnb|
|Bestandteile||Reagent (Quantity ): Assay plate (1), Standard (2), Sample Diluent (1 × 20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1 × 120μl), Detection Reagent B (1 × 120μl), Wash Buffer (25 x concentrate) (1 × 30ml), Substrate (1x10ml), Stop Solution (1 x 10ml)|
AntigendetailsProduktdetails PPP3R1 ELISA Kit Anwendungsinformationen Handhabung Bilder zurück nach oben
|Andere Bezeichnung||Ppp3r1 (PPP3R1 ELISA Kit Abstract)|
|Hintergrund||Calcineurin (CaN) is a protein phosphatase also known as protein phosphatase 2B (PP2B). Calcineurin is responsible for activating the transcription of interleukin 2 (IL-2), that stimulates the growth and differentiation of T cell response. In immunosuppressive therapy it is inhibited by cyclosporin, pimecrolimus (Elidel) and tacrolimus (FK506) - these drugs are known as calcineurin inhibitors. Calcineurin dephosphorylates NFATc, a transcription factor that can then go into the nucleus and turn on genes involved in IL-2 synthesis. When T-helper cell's receptor interacts with an antigen, the intracellular concentration of calcium in the cell rises. This increase activates calcineurin, by binding a regulatory subunit and activating calmodulin binding. Calcineurin induces different transcription factors (NF-ATs) that are important in the transcription of IL-2 genes. IL-2 activates T-helper lymphocytes and induces the production of other cytokines. In this way, it governs the action of cytotoxic lymphocytes and NK cells. The amount of IL-2 being produced by the T-helper cells is believed to influence the extent of the immune response significantly. Calcineurin is linked to receptors for two brain chemicals, NMDA and dopamine. A MIT experiment with genetically altered mice who could not produce calcineurin showed similar symptoms as in humans with schizophrenia: impairment in working memory, attention deficits, aberrant social behavior and several other abnormalities characteristic of schizophrenia. Scientist believe that calcineurin might prove to be one of the two keys, along with NFAT, in improving the function of diabetics' pancreatic beta cells.|
|Pathways||RTK Signalweg, WNT Signalweg, Fc-epsilon Rezeptor Signalübertragung, Protein targeting to Nucleus|
AnwendungsinformationenProduktdetails PPP3R1 ELISA Kit Antigendetails Handhabung Bilder zurück nach oben
|Protokoll||This assay employs the quantitative sandwich enzyme immunoassay technique. A antibody specific for CaN has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any CaN present is bound by the immobilized antibody. An enzyme-linked antibody specific for CaN is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of CaN bound in the initial step. The color development is stopped and the intensity of the color is 2 measured.|
|Aufbereitung der Reagenzien||Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer. 3 Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 10,000 pg/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (10,000 pg/mL). The Sample Diluent serves as the zero standard (0 pg/mL). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.|
|Probennahme||Cell culture supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay.|
Allow all reagents to reach room temperature. Arrange and label required number of strips.
1. Prepare all reagents, working standards and samples as directed in the previous sections.
2. Add 100 uL of Standard, Control, or sample* per well. Cover with the adhesive strip. Incubate for 2 hours at 37 °C.
3. Remove the liquid of each well, don’t wash.
4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) 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.
6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 °C.
7. Repeat the aspiration/wash as in step
5. 8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Important Note:1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
3. Duplication of all standards and specimens, although not required, is recommended.
4. When mixing or reconstituting protein solutions, always avoid foaming.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
|Ergebnisberechnung||Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. 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 CaN 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.|
|Beschränkungen||Nur für Forschungszwecke einsetzbar|
HandhabungProduktdetails PPP3R1 ELISA Kit Antigendetails Anwendungsinformationen Bilder zurück nach oben
1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay 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.
4. This assay is designed to eliminate interference by soluble receptors, ligands, 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.
|Lagerung||4 °C/-20 °C|
|Informationen zur Lagerung||The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.|