title: Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting.
abstract: SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin's transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting.
authors: Department of Sciences and Mathematics, Mississippi University for Women, Columbus, Mississippi 39701, USA;; College of Medicine, The University of Tennessee Health Sciences Center, Memphis, Tennessee 38163, USA; and.
authors: Ghanshyam D
authors: GD
authors: Heda
authors: Department of Sciences and Mathematics, Mississippi University for Women, Columbus, Mississippi 39701, USA;; Department of Biology, University of Kentucky, Lexington, Kentucky 40506, USA.
authors: Oluwabukola B
authors: OB
authors: Omotola
authors: Department of Sciences and Mathematics, Mississippi University for Women, Columbus, Mississippi 39701, USA;; College of Medicine, The University of Tennessee Health Sciences Center, Memphis, Tennessee 38163, USA; and.
authors: Rajiv P
authors: RP
authors: Heda
authors: Department of Sciences and Mathematics, Mississippi University for Women, Columbus, Mississippi 39701, USA;
authors: Jamie
authors: J
authors: Avery
mesh_terms: D015153:Blotting, Western / Q000379:methods*; D002021:Buffers; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D059027:Recycling
chemical_list: D002021:Buffers; C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: SDS-PAGE; Towbin’s transfer buffer; immunoblotting