Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening
- Karolinska Institutet, Department of Medicine, Karolinska Hospital, 171 76 Stockholm (Sweden)
- Karolinska Institutet, Department of Woman and Child Health, Karolinska Hospital, 171 76 Stockholm (Sweden)
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.
- OSTI ID:
- 20857956
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 352, 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); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
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