Western Blot is a widely used immunological technique that involves the detection of specific proteins using antigen-antibody interactions. The process begins with the separation of proteins through SDS-PAGE gel electrophoresis, followed by their transfer onto a solid support such as a nitrocellulose or PVDF membrane. Once transferred, the membrane is probed with specific antibodies that recognize and bind to the target protein. This binding is then visualized using either chemiluminescence or colorimetric detection methods, allowing for the identification and quantification of the protein of interest.
The technique combines the high resolution of SDS-PAGE with the specificity and sensitivity of immunoassays, making it a powerful tool in molecular biology. While Western blot is considered semi-quantitative, it requires careful calibration and reference standards to ensure reliable results. It differs from fully quantitative methods, as it provides relative rather than absolute measurements.
**Technical Process:**
1. **Sample Preparation:** Choose an appropriate method for extracting proteins based on the subcellular localization (nucleus, cytoplasm, cell membrane) and the nature of the target protein (denatured or native).
2. **Protein Quantification:** Use assays like Bradford or BCA to determine the concentration of the extracted protein.
3. **Gel Preparation:** Prepare an SDS-polyacrylamide gel with the appropriate acrylamide concentration depending on the size of the target protein.
4. **Electrophoresis:** Run the gel at a suitable voltage and time to separate the proteins effectively.
5. **Transfer:** Transfer the separated proteins onto a membrane using an electric field, adjusting the current and transfer time according to the gel size and protein characteristics.
6. **Blocking:** Block non-specific binding sites on the membrane using solutions like skim milk or BSA.
7. **Primary Antibody Incubation:** Incubate the membrane with a primary antibody that specifically recognizes the target protein.
8. **Washing:** Wash the membrane to remove unbound antibodies.
9. **Secondary Antibody Incubation:** Apply a secondary antibody conjugated with an enzyme or fluorescent tag for detection.
10. **Detection:** Expose the membrane to a chemiluminescent substrate or develop it using a colorimetric reagent to visualize the protein bands.
**Sample Handling Guidelines:**
- **Sample Type:** Fresh tissue or cultured cells are preferred.
- **Minimum Sample Size:** At least 1×10ⶠcells for cell samples and 30 mg for tissue samples. Providing more samples ensures better processing.
- **Storage Conditions:**
- Fresh tissues should be rapidly frozen.
- Suspended cells should be washed with cold PBS, resuspended, and stored at -80°C.
- Adherent cells should be detached using trypsin, washed, and stored at -80°C. For sensitive cells, scraping may be used instead of trypsin.
- **Communication:** Inform us in advance about the species, number of samples, and target proteins to be tested.
- **Shipping:** Send samples in ice packs or dry ice, using express delivery services like SF Express or ZTO for fast and safe transport.
**Report Contents:**
- Original exposure images of the target protein.
- Digital images of the target protein and internal controls.
- Gray-scale analysis data.
- Protein concentration data.
- Full experimental report including methodology, parameters, and instrument details.
**Pricing and Timeline:**
The cost and service duration depend on the sample type and quantity. For detailed information, please contact our customer service team or refer to your signed cooperation agreement.
**Quality Assurance:**
Our laboratory is equipped with advanced instruments and staffed by experienced scientists who have been performing Western blot experiments for years. We ensure accurate and reliable results for all our clients.
**After-Sales Support:**
We conduct regular follow-ups to improve our service quality. Details are outlined in the cooperation contract, which may vary depending on the agreement.
**Instrument and Reagents Required:**
- **Instruments:** Pressure cooker, glass homogenizer, high-speed centrifuge, spectrophotometer, -20°C refrigerator, vertical electrophoresis transfer unit, constant temperature shaker, multi-functional bleaching shaker.
- **Reagents:** Single detergent lysis buffer, 0.01 M PBS (pH 7.3), 10% separating gel, 4% stacking gel, Coomassie brilliant blue, 0.15 M NaCl solution, 2X/5X SDS loading buffer, electrophoresis buffer, transfer buffer, Lichun red dye, blocking solution, TBST, TBS, elution buffer, developer, fixer, primary and secondary antibodies, chemiluminescent reagent.
- **Consumables:** Pipette tips, centrifuge tubes, beakers, measuring cylinders, nitrocellulose membranes, gloves, plastic wrap, enamel plates, X-ray clips, X-ray films, glass rods, timers, filter paper.
Western blotting remains one of the most popular techniques in protein analysis due to its simplicity, sensitivity, and versatility. Join us at Shanghai Jinma Project Classroom and learn how to master this essential technique. Whether you're a beginner or an expert, we’re here to help you succeed.
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