Protocol for detecting and quantifying hATF4-HA in non-stress versus stress conditions using automated and quantitative Jess western blotting

Abstract

Summary: Activating transcription factor 4 (ATF4) is a key player in the integrated stress response, whose expression is subject to tight translational control. Studying its stress-provoked induction, accompanied by the general translational shutdown, is intricate because the expression of reference genes declines rapidly, and finding appropriate normalization controls is challenging. We present a protocol for human hemagglutinin-tagged ATF4 (hATF4-HA) detection and high-throughput quantification in non-stress versus stress conditions using automated and quantitative western blotting. We describe steps for seeding cells, transfecting plasmids, thapsigargin treatment, sample preparation, and target protein detection.For complete details on the use and execution of this protocol, please refer to Smirnova et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• cell culture
• gene expression
• protein expression and purification