Chapter Five: Co-localization/FRET/BRET
Contents Page Co-localization overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17 Co-localization assay formats
Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
FRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
BRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 When to u co-localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 Reagent requirements for immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 Reagent requirements for FRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 Reagent requirements for BRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
Co-localization overview
bored的意思Methods such as co-immunoprecipitation and pull-downs require the preparation of cell extracts which may not prerve the physiological conditions under which proteins may interact in a true cellular envir
onment. Using various co-localization techniques protein:protein interactions may be characterized directly in the cell without the need to create cell lysates or
isolate complexes from a cell.
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Co-localization assay formats
Immunofluorescence
Immunofluorescence can be ud to
determine whether two proteins share the same location in a cell. Two primary
antibodies that recognize the two specific proteins are added simultaneously to the sample. Two condary antibodies with
different fluorescent tags are then added. The sample is then analyzed under a confocal microscope to determine whether the two fluorescent signals overlap. If both fluorescent signals are in the same location then the proteins are located in the same cellular
region. This is an indirect way of determining whether two proteins may interact; if they are located in the same region there is a
李阳发音宝典possibility that they may bind to each other.When available, immunofluorescent antibodies to endogenously expresd proteins should be ud. However, when antibodies are not available, or when the cell line does not express the protein at sufficient levels for immunofluorescence detection, cells may be transfected with recombinant vectors
encoding tagged proteins. Antibodies to the tag can then be ud to localize the protein in the cell.
FRET
Fluorescence Resonance Energy Transfer (FRET, Figure 9) can be ud to measure interactions between two proteins in vivo.Using this technique, two different fluorescent molecules (donor and acceptor fluorophores)are ud to label the suspected protein partners. FRET is obrved by exciting the sample at the donor excitation wavelength and measuring fluorescence intensities
emitted at the wavelengths corresponding to the emission peaks of the donor compared to tho of the acceptor. When the donor and acceptor are in clo proximity (1-10nm) due to the interaction of the two proteins, the acceptor emission is predominantly obrved becau of the intermolecular FRET from the donor to the acceptor.
Another option to labeling proteins with fluorescent dyes is to express both partners as fusion proteins with different GFP (green fluorescent protein) tags. The most popular FRET pair for biological u is a cyan
fluorescent protein (CFP)-yellow fluorescent protein (YFP) pair. Both are color variants of GFP .
REFERENCES
Key original reference for FRET
1.Gordon, G. et al. (1998)Biophys. J. 74, 2702–13.
Key original reference for BRET
1.Xu, Y. et al. (1999) Proc. Natl.Acad. Sci. 96, 151–56.
U of immunofluorescence
1.Snabaitis, A. et al. (2006)
J. Biol. Chem. 281, 20252–62.2.Vandermoere, F . et al. (2006) J.Biol. Chem. 281, 14307–13.3.Burgess, A. et al. (2006) J. Gen. Virol. 87, 789–93.
U of FRET
1.Sohn, H-W. et al. (2006) Proc.Natl. Acad. Sci. 103, 8143–48.
2.Treanor, B. et al. (2006) J. Cell. Biol. 174, 153–61.
3.Hunger, K. et al. (2006) J. Bact. 188, 240–8.
4.Herrick-Davis, K. (2006)
J. Biol. Chem. 281, 27109–16.5.Kramer, J. et al. (2006) J. Immunol. 176, 711–15.
6640M A
Figure 9.Overview of FRET for the analysis of protein:protein interactions.Protein partner A is cloned into a vector containing cyan fluorescent protein (CFP ,donor).The cond partner B is cloned into a vector containing a yellow fluorescent protein (YFP ,acceptor).The vectors should also contain the appropriate elements for protein expression in mammalian cells.Both recombinant vectors are then trans-fected into mammalian cells.FRET can be obrved by exciting the sample at the donor excitation wavelength while measuring
fluorescence intensities emitted at the wavelengths corresponding to the emission peaks of the dono
r compared to that of the acceptor.When the donor and acceptor are in clo proximity,acceptor emission is predominantly obrved becau of the intermolecular FRET from the donor to the acceptor.
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REFERENCES (CONTINUED)
U of BRET
1.Shimizu, T . et al. (2006) Pro.Natl. Acad. Sci. 103, 2093–97.
2.Whittard, J. et al. (2006)
Mol. Biol. Cell 17, 2696–706.3.Goin, J. et al. (2006) J. Biol.Chem. 281, 5416–25.4.Rebois, R-V . et al. (2006) J. Cell. Sci. 119, 2807–18.5.Koshimizu, T . et al. (2006) Mol.Pharmol. 69, 1588–98.6.Sirokmany, G. et al. (2006) J. Biol. Chem. 281, 6096–105.
BRET
Bioluminescence Resonance Energy Transfer (BRET) involves the transfer of energy from a donor enzyme to suitable acceptor molecule.Using this method one protein partner is
expresd as a fusion with GFP and the other is expresd as fusion with Renilla . BRET technology is bad on the transfer of
resonant energy from a bioluminescent donor protein to a fluorescent acceptor protein using Renilla lucifera (R luc) as the donor and a GFP mutant as the acceptor molecule. BRET technology is analogous to FRET, but eliminates the need for an excitation light source and its associated problems such as high background caud by autofluorescence.
摘要英文When to u co-localization
Co-localization is ud in conjunction with other techniques to characterize protein:protein interactions in a true
mammalian environment. This technique is typically not ud for screening large numbers of prey proteins.
非行Reagent requirements for immunofluorescence •Confocal lar scanning microscope (and appropriate filters)•Tissue culture equipment •Mammalian cells •Transfection reagents
•Fluorescently labeled antibodies
Reagent requirements for FRET
•Confocal lar scanning microscope (and appropriate filters)•Tissue culture equipment •Mammalian cells
•Transfection reagents •Fluorescent dyes
•Mammalian expression vectors
containing donor tag (e.g., CFP) and coding quences for one protein partner
•Mammalian expression vectors
containing acceptor tag (e.g., YFP) and coding quences for the other protein partner
Reagent requirements for BRET
•Microplate reader capable of
luminescent and fluorescent detection •Tissue culture equipment •Mammalian cells
•Transfection reagents
•Mammalian expression vectors
containing donor tag (e.g., Renilla ) and coding quences for one protein partner
•Mammalian expression vectors
containing acceptor tag (e.g., YFP) and coding quences for the other protein水浒传英语
ico是什么
partner
relationships
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