The Csk/Src molecular C-terminal kinase (C-src) ELISA kit is designed to detect the presence of C-src using a double-antigen one-step sandwich ELISA method. The process begins by adding the sample, standard, and HRP-labeled detection antibody to microwells pre-coated with the C-src capture antigen. After incubation, the plate is washed thoroughly to remove unbound components. A TMB substrate is then added, which turns blue in the presence of peroxidase and changes to yellow when an acid is introduced. The intensity of the color produced is directly proportional to the concentration of C-src in the sample. The optical density (OD) at 450 nm is measured using a microplate reader, allowing for accurate quantification of the target protein.
**Sample Collection, Handling, and Preservation:**
1. **Serum:** Collect blood in pyrogen-free tubes. Centrifuge at 3000 rpm for 10 minutes to separate serum from red blood cells.
2. **Plasma:** Use anticoagulants such as EDTA, citrate, or heparin. Centrifuge at 3000 rpm for 30 minutes and collect the supernatant.
3. **Cell Supernatant:** Centrifuge at 3000 rpm for 10 minutes to remove debris and particulates.
4. **Tissue Homogenate:** Homogenize tissue in physiological saline and centrifuge at 3000 rpm for 10 minutes to obtain the supernatant.
5. **Storage:** If not tested immediately, aliquot samples and store at -20°C. 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)
**Operation Precautions:**
- Store the kit at 2–8°C. Allow it to equilibrate at room temperature for 20 minutes before use.
- If the washing buffer crystallizes after refrigeration, warm it gently in a water bath before use.
- Unused strips should be returned to the sealed bag and stored at low temperature.
- Do not dilute the pre-treated samples; add 10 μL directly.
- Follow the incubation time, volume, and sequence strictly as specified.
- Shake all reagents well before use.
**Kit Components:**
- Microporous plates (96-well or 48-well configurations)
- Standard solutions (1000 U/L), diluted standards
- Sample diluent, HRP-labeled detection antibody
- 20× wash buffer, TMB substrates A and B, stop solution
- Seal film, self-sealing bags, and instruction manual
**Reagent Preparation:**
- Dilute 20× wash buffer with distilled water in a 1:20 ratio.
**Washing Method:**
- Manual: Wash 5 times by filling each well with buffer, letting it sit for 1 minute, and discarding the liquid.
- Automatic: Use 350 μL of buffer per well, soak for 1 minute, and repeat 5 times.
**Procedure:**
1. Remove the required number of wells from the foil pouch and allow them to equilibrate at room temperature.
2. Set up standard, sample, and blank wells. Add 50 μL of standard solutions to the standard wells.
3. Add 10 μL of sample and 40 μL of diluent to the sample wells.
4. Add 50 μL of HRP-labeled detection antibody to each well. Seal and incubate at 37°C for 60 minutes.
5. Wash the plate 5 times, either manually or using an automatic washer.
6. Add 50 μL of TMB A and B to each well and incubate in the dark for 15 minutes.
7. Stop the reaction by adding 50 μL of stop solution and measure OD at 450 nm within 15 minutes.
**Result Analysis:**
Plot the standard curve in Excel using standard concentrations on the x-axis and corresponding OD values on the y-axis. Use the regression equation to calculate the sample concentration.
**Kit Performance:**
- Accuracy: R ≥ 0.9900
- Sensitivity: <1.0 U/L
- Specificity: No cross-reactivity with similar proteins
- Repeatability: CV <15% between and within plates
- Storage: 2–8°C, away from light and moisture
- Shelf Life: 6 months
- Detection Range: 31.2–1000 U/L
**Disclaimer:**
This kit is for research purposes only. It must not be used in clinical trials or human experiments. The company is not responsible for any misuse or deviation from instructions. Always follow the protocol carefully and do not mix reagents from different batches. Any errors caused by improper handling are the responsibility of the user.
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