Mouse NOD-like Receptor-2 (NOD-2) ELISA Kit

The NOD-like receptor-2 (NOD-2) ELISA kit is based on a double-antibody one-step sandwich ELISA method. The microwells are pre-coated with mouse NOD-2 capture antibodies. After adding the samples, standards, and HRP-conjugated detection antibodies in sequence, the plate is incubated, washed thoroughly, and then developed using TMB substrate. Under the action of horseradish peroxidase, TMB turns blue and then yellow when an acidic stop solution is added. The intensity of the color is directly proportional to the concentration of NOD-2 in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, allowing for accurate quantification of the target protein. Sample collection and handling procedures are crucial for reliable results. For serum, blood should be collected in endotoxin-free tubes, centrifuged at 3000 rpm for 10 minutes, and the supernatant carefully separated. Plasma samples should be collected using anticoagulants like EDTA, citrate, or heparin, followed by centrifugation at 3000 rpm for 30 minutes. Cell culture supernatants require centrifugation at 3000 rpm for 10 minutes to remove debris. Tissue homogenates are prepared by chopping the tissue in saline, followed by centrifugation at 3000 rpm for 10 minutes. All samples should be aliquoted, stored at -20°C, and avoid repeated freeze-thaw cycles. Thawing should be done at room temperature, ensuring even thawing before testing. For optimal performance, the following equipment is required: a microplate reader set at 450 nm, precision pipettes (0.5–10 µL, 2–20 µL, 20–200 µL, 200–1000 µL), and a 37°C incubator. Before use, the kit should be allowed to equilibrate at room temperature for 20 minutes. If the washing buffer crystallizes after refrigeration, it should be warmed gently in a water bath before use. Unused strips should be returned to the ziplock bag immediately and stored under low-temperature, dry conditions. No dilution is needed for pre-treated samples; 10 µL of the sample can be directly added. All steps must be strictly followed as outlined in the instructions. Ensure that all reagents are well mixed before use. Incubation times and volumes must be accurately adhered to for consistent results. Washing is critical and should be performed at least five times, either manually or using an automated washer. The kit includes 96-well or 48-well configurations, with pre-coated plates, standard solutions, detection antibodies, wash buffer, substrates, and stop solution. Standards are diluted to concentrations ranging from 200 ng/mL down to 0 ng/mL. The 20× wash buffer should be diluted 1:20 with distilled water before use. The procedure involves removing the required strips from the foil pouch, setting up standard, sample, and blank wells, and adding the appropriate volumes of standards, samples, and detection antibodies. After incubation at 37°C for 60 minutes, the plate is washed five times, and the substrate is added for 15 minutes in the dark. A stop solution is then added, and the OD values are read within 15 minutes. To analyze the results, a standard curve is plotted in Excel, with standard concentrations on the x-axis and corresponding OD values on the y-axis. The sample concentration is calculated using the regression equation derived from the curve. The kit offers high accuracy, with a correlation coefficient (R) of ≥0.9900, and a sensitivity of less than 1.0 ng/mL. It exhibits excellent specificity, with no cross-reactivity to other similar proteins. Repeatability is ensured with a coefficient of variation <15% between plates. The kit should be stored at 2–8°C, away from light and moisture, and has a shelf life of six months. This ELISA kit is intended for research purposes only and is not suitable for clinical trials or in vivo experiments. Any misuse or deviation from the instructions will result in responsibility falling on the user. Do not mix different batch numbers, and always follow the provided guidelines to ensure accurate and reproducible results.

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