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