Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Fluorescence Resonance Energy Transfer (FRET) measurements

MK Misun Kwak
CH Chansik Hong
JM Jongyun Myeong
EP Eunice Yon June Park
JJ Ju-Hong Jeon
IS Insuk So
request Request a Protocol
ask Ask a question
Favorite

Three FRET images (cube settings for CFP, YFP, and Raw FRET) were obtained from a pE-1 Main Unit to 3 FRET cubes (excitation, dichroic mirror, filter) via a fixed collimator. The excitation LED and the filter were sequentially rotated, and the rotation period for each filter cube was ~0.5 s. All of the images were obtained within 1.5 seconds. Each image was captured on a cooled 10 MHz (14 bit) CCD camera (ANDOR technology, USA) with 100 ms of exposure time with 2 × 2 binning (645 × 519 pixels). Using IX70, an Olympus microscope equipped with a 60× oil objective, the three-cube FRET efficiency was analyzed using MetaMorph 7.6 software (Molecular Devices, USA). For details of FRET ratio and FRET efficiency computation, please see the supplementary materials.

Copyright and License information: The Author(s) ©2018
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

0/150

tip Tips for asking effective questions

+ Description

Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.

post Post a Question
0 Q&A