Considerations when quantitating protein abundance by immunoblot

Am J Physiol Cell Physiol. 2015 Mar 15;308(6):C426-33. doi: 10.1152/ajpcell.00400.2014. Epub 2014 Dec 24.


The development of the immunoblot to detect and characterize a protein with an antisera, even in a crude mixture, was a breakthrough with wide-ranging and unpredictable applications across physiology and medicine. Initially, this technique was viewed as a tool for qualitative, not quantitative, analyses of proteins because of the high number of variables between sample preparation and detection with antibodies. Nonetheless, as the immunoblot method was streamlined and improved, investigators pushed it to quantitate protein abundance in unpurified samples as a function of treatment, genotype, or pathology. This short review, geared at investigators, reviewers, and critical readers, presents a set of issues that are of critical importance for quantitative analysis of protein abundance: 1) Consider whether tissue samples are of equivalent integrity and assess how handling between collection and assay influences the apparent relative abundance. 2) Establish the specificity of the antiserum for the protein of interest by providing clear images, molecular weight markers, positive and negative controls, and vendor details. 3) Provide convincing evidence for linearity of the detection system by assessing signal density as a function of sample loaded. 4) Recognize that loading control proteins are rarely in the same linear range of detection as the protein of interest; consider protein staining of the gel or blot. In summary, with careful attention to sample integrity, antibody specificity, linearity of the detection system, and acceptable loading controls, investigators can implement quantitative immunoblots to convincingly assess protein abundance in their samples.

Keywords: Western blot; antibody-antigen; immunoblot; immunodetection; quantitation.

Publication types

  • Research Support, N.I.H., Extramural
  • Review

MeSH terms

  • Animals
  • Antibody Specificity
  • Biomarkers / analysis
  • Blotting, Western* / standards
  • Calibration
  • Humans
  • Linear Models
  • Proteins / analysis*
  • Proteins / immunology
  • Reference Standards
  • Reproducibility of Results
  • Specimen Handling


  • Biomarkers
  • Proteins