Difference between Western and ELISA experiments - Database & Sql Blog Articles

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Western blot (also known as Western blotting) is a widely used technique in molecular biology, biochemistry, and immunogenetics. This method allows researchers to detect specific proteins within a complex mixture by utilizing antibodies that bind specifically to the target protein. After electrophoresis separates the proteins based on size, they are transferred onto a membrane where the antibody binds to its corresponding antigen. The signal is then visualized using a detection system, providing valuable information about the presence, size, and abundance of the target protein.

For sample preparation in Western blot, it is essential to first add a protein lysate containing protease inhibitors. The tissue or cell sample should be ground on ice, collected in an Eppendorf tube, and incubated on ice for 30 minutes to ensure complete lysis. After centrifugation at 4°C, 12,000 rpm for 15 minutes, the supernatant is collected as the protein sample. It is recommended to perform electrophoresis immediately after preparation, and any excess sample should be stored at -80°C for future use.

Enzyme-linked immunosorbent assay (ELISA) is a fundamental technique in immunology used to detect and quantify soluble antigens or antibodies. This method relies on the specific binding between antigens and antibodies, with the detection process often involving an enzyme-labeled secondary antibody. ELISA is highly sensitive and can be performed in various formats, such as direct, indirect, sandwich, and competitive assays. It is commonly used in diagnostic testing, research, and drug development.

When preparing samples for ELISA, brain tissue should be homogenized on ice and collected in an Eppendorf tube. Following the same centrifugation procedure as described above, each sample should be aliquoted into volumes of approximately 220–250 µL. Since ELISA typically requires 100 µL per well and duplicate measurements are standard, it's important to ensure that each sample contains at least 200 µL to avoid insufficient volume. Any leftover sample should be stored at -80°C to prevent repeated freeze-thaw cycles. Before conducting the full experiment, it's advisable to perform a preliminary test with a small number of samples or refer to literature to determine the appropriate kit and concentration range that matches the expected protein levels in the tissue. This helps avoid issues such as high concentrations exceeding the detection limit or low concentrations falling below the minimum measurable value of the kit.