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Cell lysate preparation for western blot9/14/2023 ![]() ![]() Protease and phosphatase inhibitors: As soon as lysis occurs. To analyze proteins by immunoblotting, it is necessary to bring the proteins from the sample into soluble form using the buffers, salts, and detergents compatible with the gel electrophoresis procedure. We use RIPA buffer (beyotime P0013B) for whole cell extracts and membrane-bound proteins. To avoid under- or overloading samples, determine the protein concentration of each sample prior to electrophoresis with a compatible protein assay. In this protocol, adherent cells are grown in tissue culture dishes until ready to be used for protein sample preparation.Agitate the contents in microcentrifuge tubes for 30 min at 4 ☌. Collect the cells in microcentrifuge tubes. To reduce and denature: Boil each cell lysate in sample buffer at 100C for 5 minutes and aliquot. Scrape the cells using cold plastic cell scraper. Discard the PBS, add ice-cold lysis buffer. However, if one cannot change the gel electrophoresis chemistry system, one may need to perform sample clean-up to render the sample compatible with the given system. Discard the medium in culture dishes with cells and wash the cells using ice-cold PBS. Homogenize using Qiagen Tissue Lyser (3 min at 25Hz for WAT/Liver and 5 min at 50 Hz for Muscle). Add a stainless steel bead and keep tissues on ice. Selecting a gel electrophoresis chemistry that is compatible with the buffer one’s sample is prepared in, is the simplest route. 1, Wash cells in the tissue culture flask or dish by adding cold phosphate buffered saline (PBS) and rocking gently. Add 20 uL/mg of RIPA (keep on ice) or other buffer to tissue (400-1000 uL) for liver and muscle. Avoid repeated freeze/thaw cycles of cell lysate and Western. Some buffer components may interfere with the chosen gel electrophoresis chemistry system (e.g., Tris-glycine, Bis-Tris) and cause a variety of artifacts when running the gel. Add/increase concentration of protease/phosphatase inhibitors during cell lysate preparation.To prevent these negative effects, protease and phosphatase inhibitors should be added to the lysis reagents. Cell lysis disrupts cell membranes and organelles, resulting in unregulated enzymatic activity that can reduce protein yield and lead to degraded proteins.To minimize sample variability, keep sample preparation workflows simple, and use reagents optimized for the specific sample type and target proteins. ![]()
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