Physiology 3140A Lecture 1: Toolbox 2_ Western Blot
12 Oct 2022
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We must isolate the protein from the sample. If desired protein to analyze is intracellular, Cells must be ruptured to release proteins with the use of a detergent solution. If the protein of interest is extracellular. Just grinding the tissue using a homogenizer is completed. Do this by keeping samples cold, as proteases don"t work well at low temp (protease break down proteins) Procedure: cell line of tissue grown in growth media (contains all factors for cells to grow, remove growth media. Since it contains supplied proteins (for cel growth) and any proteins excreted by the cell: wash with saline solution, remove wash buffer, add detergent to cell. Ruptures cell membranes and releases cell contents. When you rupture cell membrane you release protease which degrade proteins, and lowering temp stops protease function: scrape cells from surface of the cell culture plastic. Use a sterile cell scraper: transfer cell lysate with proteins to tube.
