The Csk/Src molecular C-terminal kinase (C-src) ELISA kit is designed to detect the presence of C-src in biological samples 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 capture antigen. After incubation, the plate is washed thoroughly to remove unbound components. A TMB substrate is then added, which turns blue under peroxidase activity and changes to yellow when an acid is introduced. The intensity of the color 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 and Preparation:**
1. **Serum:** Collect blood in endotoxin-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 to obtain plasma.
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:** If not tested immediately, aliquot the sample and store at -20°C. Avoid repeated freeze-thaw cycles. Thaw at room temperature before use.
**Required Equipment:**
- Microplate reader (450 nm)
- High-precision pipettes (0.5–10 µL, 2–20 µL, 20–200 µL, 200–1000 µL)
- Incubator set at 37°C
**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, gently warm it in a water bath before use.
- Unused strips should be returned to the sealed 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:**
- Microporous plates (12 wells × 8 or 4 strips)
- Standards (1000 U/L), diluted standards
- Sample diluent
- Detection antibody-HRP
- 20× Wash buffer
- Substrate A and B
- Stop solution
- Seal film
**Reagent Preparation:**
- Dilute 20× wash buffer with distilled water at a 1:20 ratio.
**Washing Procedure:**
- Manual: Wash 5 times by filling each well with wash buffer, letting it sit for 1 minute, then draining and patting dry.
- Automatic: Use 350 µL of wash buffer per well, soak for 1 minute, and repeat 5 times.
**Procedure Steps:**
1. Remove the required strips from the foil pouch after 20 minutes at room temperature.
2. Set up standard, sample, and blank wells.
3. Add 50 µL of standard and 10 µL of sample plus 40 µL diluent.
4. Add 50 µL of HRP-labeled antibody to each well, seal, and incubate at 37°C for 60 minutes.
5. Wash 5 times.
6. Add 50 µL of TMB A and B, incubate in the dark for 15 minutes.
7. Add stop solution and measure OD at 450 nm within 15 minutes.
**Results Interpretation:**
Plot standard concentrations against their corresponding OD values in Excel to create a standard curve. Use the equation from the regression line to calculate the sample concentration.
**Kit Performance:**
- Accuracy: R ≥ 0.9900
- Sensitivity: <1.0 U/L
- Specificity: No cross-reactivity with other similar proteins
- Repeatability: CV <15% between plates
- Storage: 2–8°C, protected 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 the instructions. Always follow the protocol carefully, and do not mix different batch numbers. Any deviations are the responsibility of the user.
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