b'Protein extraction Protein assaysProtein extraction techniques vary depending on the source ofDepending on the accuracy required and the amount and purity the starting material, the location within the cell of the protein ofof the protein available, different methods are appropriate for interest, and the downstream application. Historically, mechanicaldetermining protein concentration. Colorimetric, reagent-based disruption has been used to lyse cells and tissues, but detergent- protein assay techniques have been developed that are used based solutions have more recently been developed to efficientlyby nearly every laboratory involved in protein research. Protein lyse cells and enable the separation of subcellular structuresis added to the reagent, producing a color change in proportion without requiring physical methods. However, cell lysis disruptsto the amount added. Protein concentration is determined by cell membranes and organelles resulting in unregulatedreference to a standard curve consisting of known concentrations proteolytic activity that can reduce protein yield and function.of a purified reference protein. Unfortunately, no protein assay To prevent these negative effects, protease and phosphatasemethod exists that is either perfectly specific to proteins or inhibitors can be added to the lysis reagents. Numerousuniformly sensitive to all protein types. Therefore, successful compounds have been identified and used to inactivate or blockuse of protein assays involves selecting the method that is the activities of proteases and phosphatases by reversibly ormost compatible with the samples to be analyzed, choosing an irreversibly binding to them. appropriate assay standard, and understanding and controlling the particular assumptions and limitations that remain.Protein cleanupMany detergents and salts used in protein extraction formulations may have adverse effects on protein function or stability, or may interfere with downstream analysis; therefore, it may be necessary to remove or reduce these contaminants following cell lysis, using techniques such as dialysis or desalting. In addition, if the protein sample is too dilute, it may require concentration. Dialysis facilitates the removal of small unwanted compounds from protein in solution by selective diffusion through a semipermeable membrane. Proteins that are larger than the membrane pores are retained on the side of the membrane, but small contaminants diffuse freely through the membrane and approach an equilibrium concentration.Desalting, also known as size exclusion chromatography or gel filtration, utilizes a resin with pores that are large enough for small contaminants (e.g., salts) to penetrate, but too small for the protein of interest to enter. This causes the contaminants to slow down their rate of migration, and the larger, faster proteins separate from the slower, smaller molecules.Protein concentration and diafiltration use a semipermeable membrane to separate macromolecules from low molecular weight compounds via centrifugation. During concentration, the low molecular weight solutes are forced through the membrane while the macromolecules remain on the sample side of the membrane, where they become concentrated to a smaller volume as the reagent is forced across the membrane to the opposite side.Protein sample preparation thermofisher.com/proteinbiology 13'