Flow Cytometric Analysis of FRET to Study the Interaction Between CFP- and YFP-Tagged Proteins
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Flow Cytometric Analysis of FRET to Study the Interaction Between CFP- and YFP-Tagged Proteins. David Stepensky. Classical pathway of major histocompatibility complex (MHC) class I antigen processing, loading, and presentation. Groothuis et al, Immunol Rev 2005. Objectives.

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Flow Cytometric Analysis of FRET to Study the Interaction Between CFP- and YFP-Tagged Proteins

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Flow cytometric analysis of fret to study the interaction between cfp and yfp tagged proteins

Flow Cytometric Analysis of FRET to Study the Interaction Between CFP- and YFP-Tagged Proteins

David Stepensky


Flow cytometric analysis of fret to study the interaction between cfp and yfp tagged proteins

Classical pathway of major histocompatibility complex (MHC) class I antigen processing, loading, and presentation

Groothuis et al, Immunol Rev 2005


Objectives

Objectives

to study the interactions within the MHC I loading complex using fluorescently-tagged components:

  • the kinetics and sequence of association and dissociation of the loading complex

  • effects of the individual interactions on the loading of the MHC class I molecules with peptides

Cresswell et al, Immunol Rev 2005

Approach

  • generation of fluorescently tagged components of the loading complex

  • investigation of functioning of the tagged proteins

fluorescence-based techniques (FRET, FRAP, etc.)

biochemical techniques


Fluorescence resonance energy transfer

Fluorescence Resonance Energy Transfer

transfer of excited state energy from one fluorophore to another

CFP

HC

Tapasin

CFP

YFP

Excitation & emission spectra

YFP

Tapasin

HC

CFP

YFP

CFP excitationCFP & FRET signals

YFP excitationYFP signal

extent of interaction

%FRET


Experimental setup

Experimental setup

experimental cell lines

controls

  • interaction between MHC class I HC & Tapasin

  • M553 tapasin deficient melanoma

  • stable transfection with:

  • Tapasin-YFP &

  • MHC I heavy chain-CFP

  • multiclonal cell lines

  • interaction (FRET efficiency) was measured using confocal microscope (n=15-20 cells)

FRET, %

Tapasin-YFP

Tapasin C95A-YFP

Tapasin-YFP

Tapasin C95A-YFP

Tapasin-YFP

Tapasin C95A-YFP

w/o Tapasin

w/o Tapasin

Tapasin

Tapasin-YFP

Tapasin C95A-YFP

HLA-B44-CFP

HLA-A2.1-

CFP

HLA-A2.1-YFP-CFP

HLA-A2.1-CFP

HLA-A2.1 T134K-CFP


Flow cytometric analysis of fret to study the interaction between cfp and yfp tagged proteins

Flow Cytometric Analysis of FRET

Objective:

to obtain statistically robust measurement of FRET efficiency in the studied cell lines

CFP

  • FACSAria

  • Violet laser 405 nm

FACS setup:

Excitation & emission spectra

CFP (450/40 nm)

FRET(530/30 nm)

YFP

  • Blue laser 488 nm

YFP (530/30 nm)

  • one laser at a time, sequential acquisition of the same sample

405

CFP

488

FRET/YFP


Flow cytometric analysis of fret to study the interaction between cfp and yfp tagged proteins

FACS-FRET: the raw data

Negative

control

Exper.

sample

FRET

CFP

Positive

control

positive control

experimental sample

negative control

FRET

cells

CFP

YFP


Flow cytometric analysis of fret to study the interaction between cfp and yfp tagged proteins

FACS-FRET: the results

  • FACS-FRET results are consistent with the confocal data

  • both techniques seem to quantify correctly the interaction between the constructs

experimental cell lines

controls

Tapasin-YFP

Tapasin C95A-YFP

Tapasin-YFP

Tapasin C95A-YFP

Tapasin-YFP

Tapasin C95A-YFP

w/o Tapasin

w/o Tapasin

Tapasin

Tapasin-YFP

Tapasin C95A-YFP

FRET/CFP ratio, normalized

HLA-B44-CFP

HLA-A2.1-

CFP

HLA-A2.1-YFP-CFP

HLA-A2.1-CFP

HLA-A2.1 T134K-CFP


Flow cytometric analysis of fret to study the interaction between cfp and yfp tagged proteins

FRET assessment using FACS or confocal microscope:

selected characteristics


Flow cytometric analysis of fret to study the interaction between cfp and yfp tagged proteins

Alternative setups for FACS-FRET

He et al, Cytometry Part A, 2003

Dye, Clin Appl Immunol Rev, 2005

  • FACSVantage SE

  • spatial separation of the laser lines

  • optional laser

  • nonstandard mirrors/filters

  • FACSVantage SE

  • laser tuning to 458 nm

  • simultaneous excitation of CFP & YFP

  • nonstandard mirrors/filters


Flow cytometric analysis of fret to study the interaction between cfp and yfp tagged proteins

FACS Analysis of FRET

  • simple setup

  • combination of FACS with Confocal analysis

  • possibility of cell sorting


Flow cytometric analysis of fret to study the interaction between cfp and yfp tagged proteins

Thanks

Prof. Peter Cresswell and the group

Cell Sorter Facility

Geoff LyonTom TaylorDon Foster


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