Time-Resolved Forster Resonance Energy Transfer for Monitoring Protein Phosphorylation in Cells

Abstract

Source: Padros, J. et al., Time-resolved Förster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells. J. Vis. Exp. (2021).

This video describes the time-resolved Förster resonance energy transfer or TR-FRET assay to determine endogenous protein phosphorylation with the help of donor and acceptor fluorophores tagged on antibodies specific to a test protein. This technique is designed to measure the phosphorylation of test proteins in cell lysates.

Protocol

1. Cell culture

1. Maintain cells in a humidified 37 °C/5% CO₂ incubator and culture with either DMEM supplemented with 10% fetal bovine serum (FBS) (HeLa and A431 cells) or RPMI supplemented with 15% FBS (U266B1 cells). Culture the cells until they reach 70-80% confluence, then trypsinize them and passage or use them for the assays.
   NOTE: Culture media contained phenol red. No serum starvation was conducted for any cell line prior to conducting the assays.

2. Stimulator or inhibitor titration using the two-plate assay protocol with adherent cells

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well microplate, clear, flat bottom, polystyrene, tissue culture-treated, sterile	Corning	3595	This is the plate for culturing cells when using the two-plate assay protocol. Other cell culture 96-well plates can be used
384-well microplate, white, low volume	PerkinElmer	6007290	This is the plate for TR-FRET detection when using the two- or one-plate assay protocols. Other low volume, white 384-well plates can be used
A431 cell line	ATCC	CRL-1555
Adhesive microplate seal	PerkinElmer	6050185
Dulbecco’s modified Eagle medium (DMEM)	Wisent	319-005-CL	THUNDER TR-FRET is compatible with culture medium containing phenol red
EnVision Xcite Multilabel plate reader	PerkinElmer	2104-0020A	The assays can be performed on a variety of plate readers equipped with the TR-FRET option
HeLa cell line	ATCC	CCL-2
Roswell Park Memorial Institute 1640 medium (RPMI)	Wisent	350-007-CL	THUNDER TR-FRET is compatible with culture medium containing phenol red
Trypsin/EDTA 0.05%	Wisent	325-542-CL
Ultrapure water	NA	NA	Use Milli-Q grade water (18 MΩ.cm) to dilute Lysis Buffer and Detection Buffer