Ubiquitin-Conjugating Enzyme (UBCE) ELISA Kit

Antigen: Ubiquitin-Conjugating Enzyme (UBCE)

Synonyme: Ube2d2

Reaktivität: Human

Applikation: ELISA

Zertifikate: ISO 9001:2008

Produktnummer: ABIN366882

Produktbeschreibung

Produktmerkmale: This immunoassay kit allows for the in vitro quantitative determination of human E2/UBCE concentrations in cell culture supernates, serum, plasma and other biological fluids.

Weitere Bezeichnung: E2/ubiquitin-conjugating enzyme (E2/UBCE)

Beschreibung: Ubiquitin-conjugating enzymes, also known as E2 enzymes and more rarely as ubiquitin-carrier enzymes, perform the second step in the ubiquitination reaction that targets a protein for degradation via the proteasome. The ubiquitination process covalently attaches ubiquitin, a short protein of 76 amino acids, to a lysine residue on the target protein. Once a protein has been tagged with one ubiquitin molecule, additional rounds of ubiquitination form a polyubiquitin chain that is recognized by the proteasome's 19S regulatory particle, triggering the ATP-dependent unfolding of the target protein that allows passage into the proteasome's 20S core particle, where proteases degrade the target into short peptide fragments for recycling by the cell. A ubiquitin-activating enzyme or E1 first activates the ubiquitin by covalently attaching the molecule to its active site cysteine residue. The activated ubiquitin is then transferred to an E2 cysteine. Once conjugated to ubiquitin, the E2 molecule binds one of several ubiquitin ligases or E3s via a structurally conserved binding region. The E3 molecule is responsible for binding the target protein substrate and transferring the ubiquitin from the E2 cysteine to a lysine residue on the target protein.

Spezifität: This assay recognizes recombinant and natural human E2/UBCE. 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 E2/UBCE. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for E2/UBCE and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3'5, 5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain E2/UBCE, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm 2 nm. The concentration of E2/UBCE in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protokoll: Preparation of Reagents: 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 30 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 8000 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 (8000 pg/ml). The Sample Diluent serves as the zero standard (0 pg/ml). Biotin-antibody - Dilute to the working concentration specified on the vial label using Biotin-antibody Diluent(1:100), respectively. HRP-avidin - Dilute to the working concentration specified on the vial label using HRP-avidin Diluent(1:100), respectively. Sample Collection and Storage: 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 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. Assay Procedure: Calculation of Results: 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 E2/UBCE 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. 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.

Applikationshinweise: When mixing or reconstituting protein solutions, always avoid foaming. 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. When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless to gradations of blue. Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.

Bestandteile: Reagent Quantity Assay plate 1 Standard 2 Sample Diluent 1 x 20ml Biotin-antibody Diluent 1 x 10ml HRP-avidin Diluent 1 x 10ml Biotin-antibody 1 x 120ul HRP-avidin 1 x 120ul Wash Buffer 1 x 20ml (25 x concentrate) TMB Substrate 1 x 10ml Stop Solution 1 x 10ml STORAGE Unopened test kits should be stored at 2-8°Cupon receipt and the microtiter plate should be kept in a sealed bag with desiccants 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. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Lagerung: Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants 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. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Beschränkungen: Nur für Forschungszwecke einsetzbar