Western Blot Processing Optimization: The Perfect Blot

Methods Mol Biol. 2022:2349:65-80. doi: 10.1007/978-1-0716-1585-0_4.

Abstract

Western blot processing is a well-established procedure that includes protein extraction from tissues and cells, gel electrophoresis separation, transfer to a membrane, and immunodetection with specific antibodies. Here, we show that optimization of washing helps to maximize the specific interactions of antigens and antibodies. Performing all washing steps at 4 °C ensures a maximal signal to noise ratio and reduces nonspecific signals.

Keywords: Antibodies specificity; Automation; Electrophoresis; Immunodetection; Washing temperature; Western blot.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies
  • Antigens
  • Blotting, Western*
  • Electrophoresis, Polyacrylamide Gel
  • Proteins

Substances

  • Antibodies
  • Antigens
  • Proteins