If a detergent-free lysis buffer is used, the sample should be homogenized using a Dounce homogenizer, sonicated, or passed through a 28-gauge needle. High salt concentrations can also be used to promote protein solubility, although excess salt can produce wavy bands during electrophoresis. Many proteins are soluble at a higher pH. If native lysis conditions are required, proteins may be solubilized by varying the pH and ionic strength of the lysis buffer. RIPA or perform mitochondrial fractionation. Table 3: Select an appropriate lysis buffer based on the target protein’s localization.
In some cases, chaotropic agents that disrupt hydrogen bonding, such as 8M urea, can be used to isolate difficult to solubilize proteins, however, these samples should not be heated as urea can be hydrolyzed to cyanate, which can chemically modify the protein sample. RIPA (radioimmunoprecipitation assay) buffer, which contains SDS, is well suited for preparing whole cell extracts, membrane-bound extracts, and nuclear extracts, but disrupts protein-protein interactions. Zwitterionic detergents, such as CHAPS, are better able to solubilize membrane proteins. NP-40 buffer or Triton X-100-containing buffer can be used to prepare whole cell extracts, cytoplasmic extracts, and membrane-bound extracts, however, hydrophobic proteins may remain insoluble under these conditions. The epitope information and / or suggested immunoblotting conditions should be indicated on the antibody’s data sheet.ĭistinct subcellular fractions of a biological sample can be isolated by varying the strength of the detergent in the lysis buffer (Table 3). In general, denaturing conditions are used for cell lysis, however, if the antibody only recognizes the target protein in its native state, do not use SDS in the electrophoresis procedure and do not heat the sample. The formulation of the lysis buffer will depend on the subcellular location of the protein of interest, as well as on whether the antibody’s epitope is still present on the protein once it has been denatured. A specific protein of interest is then detected on the membrane using antibodies that recognize an epitope in the target protein, horseradish peroxidase (HRP)-conjugated secondary antibodies, and a chemiluminescent-based detection substrate. The proteins are then transferred to a solid-phase membrane. The sample is then subject to gel electrophoresis allowing the proteins in the mixture to be separated according to their size. In this example, a lysate sample is prepared from cultured cells. Western blotting relies on the electrophoretic separation of proteins from a complex mixture based on their mass, the transfer of these proteins to a solid matrix, and the detection of specific proteins of interest on the matrix using antibodies.įigure 1: The flow-through of a typical western blot experiment. The western blot, also commonly known as immunoblot, has since become an essential and ubiquitous technique in biology and medical labs around the world. The research groups of George Stark at Stanford University and Harry Towbin at Friedrich Miescher Institut in Switzerland published similar immunoblotting techniques at roughly the same time. Despite this, the paper was widely circulated and eventually published in 1981. Interestingly, the manuscript detailing this technique was initially rejected, with reviewers criticizing the method’s name as “flippant and frivolous whimsy”. He subsequently termed this method the “western blot”, in a nod to its predecessors. Neal Burnette, a postdoc at the Fred Hutchinson Cancer Research Center, developed a method for visualizing proteins separated by SDS-PAGE using monoclonal antibodies. This method was quickly followed two years later by the invention of the “northern blot”, which could detect specific RNA molecules using radio-labeled DNA probes. In 1975, Edwin Southern invented the eponymously-named “southern blot”, a technique in which DNA fragments are separated through electrophoresis based on their size and then transferred to a nitrocellulose membrane for detection. In fact, essential western blot methodology, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and monoclonal antibodies with antigen specificity, were only established in 19, respectively. It may be surprising to learn that the history of the western blot only stretches back to the late 1970s.
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