Topic
Southwestern blot
About: Southwestern blot is a research topic. Over the lifetime, 287 publications have been published within this topic receiving 69157 citations. The topic is also known as: Southwestern blotting.
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TL;DR: A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets that results in quantitative transfer of ribosomal proteins from gels containing urea.
Abstract: A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
53,030 citations
TL;DR: The detection of murine leukemia virus antigens in complex cellular lysates was used to demonstrate the efficacy of this electrophoretic transfer technique.
8,346 citations
TL;DR: Evidence is presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator.
Abstract: A method, called "protein blotting," for the detection of DNA-binding proteins is described. Proteins are separated on an SDA-polyacrylamide gel. The gel is sandwiched between 2 nitrocellulose filters and the proteins allowed to diffuse out of the gel and onto the filters. The proteins are tightly bound to each filter, producing a replica of the original gel pattern. The replica is used to detect DNA-binding proteins, RNA-binding proteins or histone-binding proteins by incubation of the filter with [32P]DNA, [125I]RNA, or [125I] histone. Evidence is also presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator. Strategies for the detection of specific protein-DNA interactions are discussed.
681 citations
TL;DR: The downward alkaline capillary transfer of DNA and RNA from agarose gel to a hybridization membrane was performed using a transfer solution containing 3 M NaCl and 8 mM NaOH to allow for a hybridized efficiency higher than that of the standard blotting protocols performed at neutral pH.
548 citations
TL;DR: A method is described for the detection of concanavalin A-binding proteins in complex mixtures where the proteins are separated by gel electrophoresis and then blotted onto a nitrocellulose sheet where they form an immobilized replica of the original gel.
357 citations