Western Blot is a widely used immunological technique that allows for the detection and analysis of specific proteins within complex mixtures. The process involves separating proteins by size using SDS-PAGE gel electrophoresis, transferring them to a solid support such as a nitrocellulose membrane, and then detecting the target protein using specific antibodies. This method is highly sensitive and offers semi-quantitative results, making it an essential tool in molecular biology research.
The procedure typically begins with the isolation of total protein from the sample, which can be tissue, cells, or other biological materials. It is crucial to select the appropriate extraction method based on the location of the target protein (e.g., nucleus, cytoplasm, or cell membrane) and its biochemical properties (denatured or native). Once the protein is extracted, its concentration is determined using methods like Bradford or BCA assays to ensure accurate loading during electrophoresis.
Next, a suitable SDS-PAGE gel is prepared according to the molecular weight of the target protein. The gel is run under controlled voltage and time conditions to achieve optimal separation. After electrophoresis, the proteins are transferred onto a membrane via electroblotting, where they are then blocked to reduce non-specific binding. Primary and secondary antibodies are applied sequentially, followed by detection using chemiluminescence or colorimetric methods.
To ensure reliable results, proper sample preparation is essential. Fresh tissues or cells should be used, with a minimum cell count of 1×10ⶠfor cell samples and 30 mg for tissue samples. Tissues should be rapidly frozen, while cells must be washed with cold PBS before storage at -20°C. For adherent cells, careful trypsinization or scraping may be required depending on the nature of the target protein.
When submitting samples, it is important to provide details such as species, number of samples, and the specific proteins to be analyzed. Samples should be shipped on dry ice or ice packs, preferably via express delivery services like SF Express or ZTO.
The final report includes images of the Western blot, gray-scale analysis data, and a detailed summary of the experimental procedure, parameters, and instruments used. Our service guarantees high-quality results through advanced equipment, experienced personnel, and strict quality control measures.
In addition to the main procedure, we also offer comprehensive after-sales support, including customer follow-ups to ensure satisfaction. All services are outlined in the signed cooperation contract, and fees vary depending on the complexity of the experiment.
For those new to Western Blot, our lab provides training sessions to help you understand the entire process, from sample preparation to data interpretation. With years of experience in protein analysis, we are committed to delivering accurate and reliable results tailored to your research needs.
Equipment required includes a pressure cooker, glass homogenizer, high-speed centrifuge, spectrophotometer, low-temperature refrigerator, vertical electrophoresis system, shaker, and more. Reagents include lysis buffer, PBS, SDS loading buffer, transfer buffer, blocking solution, and various antibody reagents. Consumables such as pipette tips, centrifuge tubes, membranes, gloves, and X-ray films are also necessary for the experiment.
Western Blot remains one of the most powerful techniques in protein detection due to its sensitivity, specificity, and ease of use. Whether you're conducting basic research or clinical studies, our team is here to support you every step of the way. Join us in the Shanghai Jinma Project classroom and learn how to master this essential technique.
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