Protein purification and analysis: Next generation western blotting techniques

Manish Mishra, Shuchita Tiwari, Aldrin V Gomes

Research output: Contribution to journalReview articlepeer-review

70 Scopus citations


Introduction: Western blotting is one of the most commonly used techniques in molecular biology and proteomics. Since western blotting is a multistep protocol, variations and errors can occur at any step reducing the reliability and reproducibility of this technique. Recent reports suggest that a few key steps, such as the sample preparation method, the amount and source of primary antibody used, as well as the normalization method utilized, are critical for reproducible western blot results. Areas covered: In this review, improvements in different areas of western blotting, including protein transfer and antibody validation, are summarized. The review discusses the most advanced western blotting techniques available and highlights the relationship between next generation western blotting techniques and its clinical relevance. Expert commentary: Over the last decade significant improvements have been made in creating more sensitive, automated, and advanced techniques by optimizing various aspects of the western blot protocol. New methods such as single cell-resolution western blot, capillary electrophoresis, DigiWest, automated microfluid western blotting and microchip electrophoresis have all been developed to reduce potential problems associated with the western blotting technique. Innovative developments in instrumentation and increased sensitivity for western blots offer novel possibilities for increasing the clinical implications of western blot.

Original languageEnglish (US)
Pages (from-to)1037-1053
Number of pages17
JournalExpert Review of Proteomics
Issue number11
StatePublished - Oct 13 2017


  • Immunoblot
  • Next generation Western blotting
  • Protein purification
  • Protein sample preparation
  • Western blotting
  • Western blotting techniques

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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