Western Blotting with Fast SDS-PAGE and Semi-Dry Protein Transfer.

Autor: Garić D; Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee., Dumut DC; Program in Infectious Diseases and Immunity in Global Health, McGill University Health Center, Montreal, Quebec, Canada.; Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada., Centorame A; Program in Infectious Diseases and Immunity in Global Health, McGill University Health Center, Montreal, Quebec, Canada.; Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada., Radzioch D; Program in Infectious Diseases and Immunity in Global Health, McGill University Health Center, Montreal, Quebec, Canada.; Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada.
Jazyk: angličtina
Zdroj: Current protocols [Curr Protoc] 2023 Jul; Vol. 3 (7), pp. e833.
DOI: 10.1002/cpz1.833
Abstrakt: Western blotting is a method widely used in cell and molecular biology for the specific detection of proteins in biological samples. It is time-consuming and normally takes up to 2 days to complete. This protocol introduces a more time-efficient method to complete western blots, covering the preparation of protein extracts (including strategies for solubilization), electrophoretic separation of proteins, transfer of proteins to the membrane, and probing with antibodies. We describe an SDS-PAGE protocol that achieves a gradient-like separation of proteins (10-400 kDa) on a single-percentage polyacrylamide gel in only 45 min. Additionally, we present a rapid (10-14 min) semi-dry transfer of proteins from standard Tris/glycine polyacrylamide gels onto a membrane using homemade Tris/HEPES- or Tris/EPPS-based buffers. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Support Protocol 1: Cell lysis and protein extraction Support Protocol 2: Protein quantification with BCA assay and sample preparation for loading on gel Basic Protocol 2: Protein transfer with a fast semi-dry transfer (FSDT) buffer Basic Protocol 3: Immunoprobing, chemiluminescent visualization, stripping, and reuse of membranes.
(© 2023 Wiley Periodicals LLC.)
Databáze: MEDLINE
Externí odkaz: