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

Published: April 30, 2023

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 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. …

開示

The authors have nothing to disclose.

Materials

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

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記事を引用
Time-Resolved Forster Resonance Energy Transfer for Monitoring Protein Phosphorylation in Cells. J. Vis. Exp. (Pending Publication), e21329, doi: (2023).

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