Cytochrome P450 3A4 (CYP3A4) protein quantification using capillary western blot technology and total protein normalization

J Pharmacol Toxicol Methods. 2021 Nov-Dec:112:107117. doi: 10.1016/j.vascn.2021.107117. Epub 2021 Aug 30.

Abstract

The western blot (WB) is the predominate method for protein quantification, frequently used in pharmacological and toxicological studies. To control for technical variation, WB signals are normalized through immunodetection of an internal standard "house-keeping" gene or total protein quantification via staining of the same blot or a duplicate, sister blot. Increasing evidence suggests that house-keeping genes are subject to change after drug treatment or under disease states, causing protein quantification errors in WB. Recent advances in automated capillary-based WB technologies enable measurement of the protein of interest, internal standards, and total protein in a single capillary. Using this approach, we quantified cytochrome P450 3A4 (CYP3A4) across 179 liver samples and compared normalization by both β-actin and total protein to determine which better functions as an internal standard. CYP3A4 is responsible for metabolism of a wide array of xenobiotics and is known to exhibit large inter-person variation, making it a good candidate to evaluate protein quantification. We observed significant differences in β-actin protein levels between liver samples (~20-fold) and found better correlation between CYP3A4 protein and mRNA using total protein normalization than β-actin, indicating total protein normalization to be less error prone for estimation of CYP3A4. Furthermore, by using total protein normalization, we confirmed significant association between CYP3A4 protein expression and the functional CYP3A4 variant CYP3A4*22, which contains two linked SNPs rs35599367 and rs62471956. Our results indicate that the automatic capillary WB instrument combined with total protein normalization provides a high throughput and robust approach for protein quantification.

Keywords: CYP3A4; Cytochrome P450; Polymorphisms; Protein normalization; Western blot.

MeSH terms

  • Blotting, Western
  • Cytochrome P-450 CYP3A* / genetics
  • Humans
  • Proteins*
  • RNA, Messenger
  • Technology

Substances

  • Proteins
  • RNA, Messenger
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human