Work

Protocol for the removal of bound endotoxin from tau protein produced in Escherichia coli

Year: 2025

Type: article

Abstract: Tau is a widely studied protein that has implications for the progression of neurodegenerative disease. Here, we present a protocol for removing bound endotoxin from Escherichia coli ( E. coli )-derived tau protein and verifying endotoxin removal. We describe steps for Triton X-114 treatment, seeding of HEK-Blue hTLR4 with endotoxin standards, collecting conditioned media, and endotoxin quantification. We then detail procedures for stimulating microglia and detecting cytokine secretion. This protocol is crucial for research studies that utilize tau in cell culture. For complete details on the use and execution of this protocol, please refer to Shultz et al. 1 • Steps for easy removal of bound endotoxin from recombinantly produced tau protein • Instructions to determine endotoxin levels in tau preps using hTLR4-expressing cells • Guidance on detecting inflammatory responses in microglia after tau stimulation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Tau is a widely studied protein that has implications for the progression of neurodegenerative disease. Here, we present a protocol for removing bound endotoxin from Escherichia coli ( E. coli )-derived tau protein and verifying endotoxin removal. We describe steps for Triton X-114 treatment, seeding of HEK-Blue hTLR4 with endotoxin standards, collecting conditioned media, and endotoxin quantification. We then detail procedures for stimulating microglia and detecting cytokine secretion. This protocol is crucial for research studies that utilize tau in cell culture.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104177

Authors: Andrew Shultz, Daya Mena, Jessica L. Roy, Jonathan Nulman, Jennifer N. Rauch

Institution: University of Massachusetts Amherst

Language: English

Topic: Alzheimer's disease research and treatments

Subfield: Physiology

Field: Medicine

Domain: Health Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $1,550