Human high molecular weight cytokeratin (CK-HMW) ELISA kit

The Human High Molecular Weight Cytokeratin (CK-HMW) ELISA Kit is based on a double-antibody one-step sandwich ELISA method. The microtiter plates are pre-coated with capture antibodies specific to CK-HMW. After adding the samples, standards, and HRP-labeled detection antibodies sequentially, the plate is incubated, washed thoroughly, and then developed using TMB as the substrate. Under the action of peroxidase, TMB turns blue and then yellow when an acid is added. The intensity of the color correlates directly with the concentration of CK-HMW in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, and the sample concentration is calculated accordingly. **Sample Collection and Preparation:** 1. **Serum:** Use endotoxin-free tubes. After blood collection, centrifuge at 3000 rpm for 10 minutes to separate serum from red blood cells. 2. **Plasma:** Collect using EDTA, citrate, or heparin as anticoagulants. Centrifuge at 3000 rpm for 30 minutes and collect the supernatant. 3. **Cell Supernatant:** Centrifuge at 3000 rpm for 10 minutes to remove debris. 4. **Tissue Homogenate:** Homogenize tissue in physiological saline, then centrifuge at 3000 rpm for 10 minutes to collect the supernatant. 5. **Storage:** Store samples at -20°C if not tested immediately. Avoid repeated freeze-thaw cycles. Thaw at room temperature before use. **Required Equipment:** - Microplate reader (450 nm) - Precision pipettes (0.5–10 µL, 2–20 µL, 20–200 µL, 200–1000 µL) - 37°C incubator **Precautions:** - Store the kit at 2–8°C. Let it equilibrate at room temperature for 20 minutes before use. If the washing buffer crystallizes after removal from the fridge, warm it gently in a water bath before use. - Unused strips should be returned to the ziplock bag and stored at low temperature. - No need to dilute the sample; add 10 µL directly. - Follow the protocol strictly, including incubation time, volume, and sequence. - Shake all reagents well before use. **Kit Components:** - Microwell plates (96-well or 48-well configuration) - Standards (200 ng/mL) - Standard dilutions - Sample diluent - Detection antibody-HRP - 20× Wash buffer - Substrate A and B - Stop solution - Seal film - Instructions **Reagent Preparation:** - Dilute 20× wash buffer by mixing 1 part with 19 parts of distilled water. **Washing Procedure:** - Manual: Add washing buffer, let stand for 1 minute, discard, and repeat 5 times. - Automatic: Use 350 µL per well, soak for 1 minute, and wash 5 times. **Procedure Steps:** 1. Remove the required strips from the foil pouch after 20 minutes of equilibration. 2. Set up standard, sample, and blank wells. 3. Add 50 µL of standard solutions and 10 µL of sample plus 40 µL diluent to each sample well. 4. Add 50 µL of HRP-labeled detection antibody to each well, seal, and incubate at 37°C for 60 minutes. 5. Wash 5 times with washing buffer. 6. Add 50 µL of TMB A and B, incubate in the dark for 15 minutes. 7. Add 50 µL stop solution and measure OD at 450 nm within 15 minutes. **Result Analysis:** Plot standard concentrations against OD values in Excel to generate a standard curve. Use the regression equation to calculate sample concentrations. **Kit Performance:** - Accuracy: R ≥ 0.9900 - Sensitivity: <1.0 ng/mL - Specificity: No cross-reactivity - Repeatability: CV <15% - Storage: 2–8°C, protected from light and moisture - Shelf Life: 6 months - Detection Range: 6.25–200 ng/mL **Disclaimer:** This kit is for research purposes only. Not suitable for clinical use. The company is not responsible for misuse or deviations from instructions. Do not mix different batch numbers. Users are fully responsible for any errors.

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