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Title: Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening

Abstract

The fluorescent proteins ECFP and HcRed were shown to give an easily resolved FRET-signal when expressed as a fusion inside mammalian cells. HeLa-tat cells expressing ECFP, pHcRed, or the fusion protein pHcRed-ECFP were analyzed by flow cytometry after excitation of ECFP. Cells expressing HcRed-ECFP, or ECFP and HcRed, were mixed and FACS-sorted for FRET positive cells: HcRed-ECFP cells were greatly enriched (72 times). Next, cloned human antibodies were fused with ECFP and expressed anchored to the ER membrane. Their cognate antigens (HIV-1 gp120 or gp41) were fused to HcRed and co-expressed in the ER. An increase of 13.5 {+-} 1.5% (mean {+-} SEM) and 8.0 {+-} 0.7% in ECFP fluorescence for the specific antibodies reacting with gp120 or gp41, respectively, was noted after photobleaching. A positive control (HcRed-ECFP) gave a 14.8 {+-} 2.6% increase. Surprisingly, the unspecific antibody (anti-TT) showed 12.1 {+-} 1.1% increase, possibly because overexpression in the limited ER compartment gave false FRET signals.

Authors:
 [1];  [2];  [3];  [4]
  1. Karolinska Institutet, Department of Medicine, Karolinska Hospital, 171 76 Stockholm (Sweden)
  2. Karolinska Institutet, Department of Woman and Child Health, Karolinska Hospital, 171 76 Stockholm (Sweden)
  3. (Sweden)
  4. Karolinska Institutet, Department of Medicine, Karolinska Hospital, 171 76 Stockholm (Sweden). E-mail: mats.persson@ki.se
Publication Date:
OSTI Identifier:
20857956
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 352; Journal Issue: 2; Other Information: DOI: 10.1016/j.bbrc.2006.11.042; PII: S0006-291X(06)02502-2; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AIDS VIRUS; ANTIBODIES; ANTIGENS; EXCITATION; FLUORESCENCE; MEMBRANE PROTEINS; SCANNING ELECTRON MICROSCOPY; SCREENING

Citation Formats

Johansson, Daniel X., Brismar, Hjalmar, Royal Institute of Technology, Department of Cell Physics, KTH/SCFAB, 106 91 Stockholm, and Persson, Mats A.A.. Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2006.11.042.
Johansson, Daniel X., Brismar, Hjalmar, Royal Institute of Technology, Department of Cell Physics, KTH/SCFAB, 106 91 Stockholm, & Persson, Mats A.A.. Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening. United States. doi:10.1016/j.bbrc.2006.11.042.
Johansson, Daniel X., Brismar, Hjalmar, Royal Institute of Technology, Department of Cell Physics, KTH/SCFAB, 106 91 Stockholm, and Persson, Mats A.A.. Fri . "Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening". United States. doi:10.1016/j.bbrc.2006.11.042.
@article{osti_20857956,
title = {Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening},
author = {Johansson, Daniel X. and Brismar, Hjalmar and Royal Institute of Technology, Department of Cell Physics, KTH/SCFAB, 106 91 Stockholm and Persson, Mats A.A.},
abstractNote = {The fluorescent proteins ECFP and HcRed were shown to give an easily resolved FRET-signal when expressed as a fusion inside mammalian cells. HeLa-tat cells expressing ECFP, pHcRed, or the fusion protein pHcRed-ECFP were analyzed by flow cytometry after excitation of ECFP. Cells expressing HcRed-ECFP, or ECFP and HcRed, were mixed and FACS-sorted for FRET positive cells: HcRed-ECFP cells were greatly enriched (72 times). Next, cloned human antibodies were fused with ECFP and expressed anchored to the ER membrane. Their cognate antigens (HIV-1 gp120 or gp41) were fused to HcRed and co-expressed in the ER. An increase of 13.5 {+-} 1.5% (mean {+-} SEM) and 8.0 {+-} 0.7% in ECFP fluorescence for the specific antibodies reacting with gp120 or gp41, respectively, was noted after photobleaching. A positive control (HcRed-ECFP) gave a 14.8 {+-} 2.6% increase. Surprisingly, the unspecific antibody (anti-TT) showed 12.1 {+-} 1.1% increase, possibly because overexpression in the limited ER compartment gave false FRET signals.},
doi = {10.1016/j.bbrc.2006.11.042},
journal = {Biochemical and Biophysical Research Communications},
number = 2,
volume = 352,
place = {United States},
year = {Fri Jan 12 00:00:00 EST 2007},
month = {Fri Jan 12 00:00:00 EST 2007}
}