PDHA1 Kit ELISA

N° du produit ABIN456323

Détail du produit PDHA1 Kit ELISA

Antigène: PDHA1 (Pyruvate Dehydrogenase (Lipoamide) alpha 1 (PDHA1))

Reactivité: Souris

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Application: ELISA

Fonction: This immunoassay kit allows for the specific measurement of Mouse Pyruvate dehydrogenase-E1 concentrations in cell culture supernates, serum and plasma.

Type d'échantillon: Cell Culture Supernatant, Serum, Plasma

Analytical Method: Quantitative

Specificité: This assay recognizes recombinant and natural Mouse Pyruvate dehydrogenase-E1.

Attributs du produit: Mus musculus,Mouse,Pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial,PDHE1-A type I,Pdha1,Pdha-1,1.2.4.1

Ingrédients: Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), 2 Detection Reagent A (1x120µl), Detection Reagent B (1x120µl), Wash Buffer(25 x concentrate) (1x30ml), Substrate (1x10ml), Stop Solution (1x10ml)

Information d'application

Volume d'échantillon: 100 μL

Plaque: Pre-coated

Protocole: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Pyruvate dehydrogenase-E1 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Pyruvate dehydrogenase-E1 present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for Pyruvate dehydrogenase-E1 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 Pyruvate dehydrogenase-E1 bound in the initial step. The color development is stopped and the intensity of the color is measured.

Préparation des réactifs:

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. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 100 nmol/L. 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 (100 nmol/L). The Sample Diluent serves as the zero standard (0 nmol/L). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.

Prélèvement de l'échantillon: 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.

Procédure de l'essai:

Allow all reagents to reach room temperature. Arrange and label required number of strips. 3
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.

Calcul des résultats:

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 4 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 Pyruvate dehydrogenase-E1 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.

Restrictions: For Research Use only

Stockage

Conseil sur la manipulation: 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.

Stock: 4 °C/-20 °C

Stockage commentaire: 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.

Détail du antigène

Antigène: PDHA1 (Pyruvate Dehydrogenase (Lipoamide) alpha 1 (PDHA1))

Autre désignation: Pdha1 (PDHA1 Produits)

Synonymes: PDHA Kit ELISA, PDHCE1A Kit ELISA, PHE1A Kit ELISA, Pdha-1 Kit ELISA, pdha Kit ELISA, phe1a Kit ELISA, pdhce1a Kit ELISA, im:6895726 Kit ELISA, pdha1 Kit ELISA, wu:fd18b01 Kit ELISA, wu:fo96f03 Kit ELISA, zgc:73271 Kit ELISA, zgc:86692 Kit ELISA, Pdha1 Kit ELISA, wu:fp73b04 Kit ELISA, zgc:92705 Kit ELISA, NI36_05250 Kit ELISA, pdha1-a Kit ELISA, pdha1-b Kit ELISA, pyruvate dehydrogenase E1 alpha 1 subunit Kit ELISA, pyruvate dehydrogenase E1 alpha 1 Kit ELISA, pyruvate dehydrogenase (lipoamide) alpha 1 Kit ELISA, pyruvate dehydrogenase (lipoamide) alpha 1 pseudogene Kit ELISA, 2-oxo acid dehydrogenase Kit ELISA, pyruvate dehydrogenase (acetyl-transferring) E1 component subunit alpha Kit ELISA, pyruvate dehydrogenase alpha subunit protein Kit ELISA, pyruvate dehydrogenase (acetyl-transferring) protein, alpha subunit Kit ELISA, precursor of dehydrogenase pyruvate dehydrogenase E1 component alpha subunit Kit ELISA, pyruvate dehydrogenase Kit ELISA, pyruvate dehydrogenase E1 alpha 1 subunit a Kit ELISA, pyruvate dehydrogenase E1 alpha 1 subunit b Kit ELISA, pyruvate dehydrogenase E1 component subunit alpha Kit ELISA, pyruvate dehydrogenase (lipoamide) alpha 1 S homeolog Kit ELISA, PDHA1 Kit ELISA, Pdha1 Kit ELISA, pdha1 Kit ELISA, LOC719277 Kit ELISA, VNG_RS07485 Kit ELISA, RR_RS04695 Kit ELISA, pdhA1 Kit ELISA, pdha1a Kit ELISA, pdha1b Kit ELISA, NI36_RS05240 Kit ELISA, pdha1.S Kit ELISA

Sujet: Pyruvate dehydrogenase (E1) is the first component enzyme of pyruvate dehydrogenase complex (PDC). EC 1.2.4.1.Biochemical and structural data for E1s revealed a mechanism of activation of TPP cofactor by forming the conserved hydrogen bond with glutamate residue (Glu59 in human E1) and by imposing a V-conformation that brings the N4’ atom of the aminopyrimidine to the distance required for the intramolecular hydrogen bonding with the thiazolium C2 atom. Pyruvate Dehydrogenase is a large complex containing many copies of each of three enzymes, E1, E2, and E3. The inner core of the mammalian Pyruvate Dehydrogenase complex is an icosahedral structure consisting of 60 copies of E2. At the periphery of the complex are 30 copies of E1 (itself a tetramer with subunits a2b2) and 12 copies of E3 (a homodimer), plus 12 copies of an E3 binding protein that links E3 to E2.

Pathways: L'effet Warburg