Effect of Thermal and Cold Denaturation on ABCB1 and ATP1A1 Transmembrane Proteins

Abstract Western blot is a conventional method for the detection of proteins using specific antibodies and several experimental steps. Expression analysis of transmembrane proteins such as ABCB1 and ATP1A1 by Western blot is challenging due to target protein-specific variations in experimental procedures. The standard denaturation temperature for proteins (95°C and 5 min) interfered with the detection of ABCB1 and ATP1A1 proteins. The aim of the study was to determine the denaturation of ABCB1 and ATP1A1 proteins in HepG2 cells by Western blot at different temperatures and durations and to optimize the denaturation temperature for the best results. In addition to transmembrane proteins, phospho-AMPK-α proteins in the nucleus and cytoplasm were also analyzed. Initially, 70°C (3–10 min) was used as the lower temperature to optimize denaturation. The protein bands of ATP1A1 reached an optimal level for Western blot analysis. However, there was no change in ABCB1. The study found that optimal denaturation of transmembrane proteins occurs at ambient or cold temperatures, while higher temperatures may lead to protein aggregation or loss. We propose to consider the denaturation of transmembrane proteins at ambient and cold temperatures, although each protein needs to be confirmed separately in different cell types.