We would like to thank the Neuroscience Imaging Service at Stanford University School of Medicine for providing equipment and space for this project. This research was supported by intramural funding of the Stanford Cancer Institute and the Gynecologic Oncology Division Stanford as well as GINOP-2.3.2-15-2016-00026, GINOP-2.3.3-15-2016-00030, NN129371, ANN135107 from the National Research, Development and Innovation Office, Hungary.

1. Plasmid construction
<ol>
	<li>For generating the eGFP-mCherry1 fusion probe, use an N1 mammalian cell expression vector (see Table of Materials) with mCherry110 inserted using the restriction sites AgeI and BsrGI.</li>
	<li>Use the following oligonucleotides to amplify eGFP11 without a stop codon as SalI-BamHI fragment: N-terminal primer 5&#39;-AAT TAA CAG TCG ACG ATG GTG AGC AAG GGC GAG G 3&#39; and C-terminal primer 5&#39;...

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The authors have nothing to disclose.

The presented protocol details the use of the genetically coupled one-to-one fluorescent protein calibration probe for quantifying FRET using the detection of sensitized emission of the acceptor and quenching of the donor molecule by confocal microscopy. This method can be applied to assess protein interactions in the physiological context of the living cell in different subcellular compartments. Spatial resolution can be further improved by applying the presented algorithm to calculate FRET efficiencies in each pixel of...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62241">Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission</a>. The Authors section was updated from:
Gy&#246;rgy V&#225;mosi1 
Sarah Miller2 
Molika Sinha2 
Gabor Mocs&#225;r1 
Malte Renz2 
1Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen 
2Gynecologic Oncology Division, Stanford University School of Medicine
to:
Gy&#246;rgy V&#225;mosi1 
Sarah Miller2 
Molika Sinha2 
Maria Kristha Fernandez2 
Gabor Mocs&#225;r1 
Malte Renz2 
1Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen 
2Gynecologic Oncology Division, Stanford University School of Medicine

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62241">Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission</a>. The Authors section was updated.

Erratum: Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

Microscopy-based analysis of F&#246;rster Resonance Energy Transfer (FRET) permits assessment of interactions between proteins in live cells. It provides spatial and temporal information, including information on where in the cell and in which subcellular compartment the interaction takes place and if this interaction changes over time.
Theodor F&#246;rster laid the theoretical foundation of FRET in 19481. FRET is a radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance of the molecules and the relative orientation of their transition dipoles as well as the overlap between the donor emission and acceptor absorption spectra. The rate of energy transfer is inversely proportional to the sixth power of the donor-acceptor distance. Thus, FRET can be used to measure molecular proximity in the range of 1-10 nm.
FRET competes with other de-excitation processes of the donor molecule and results in the so-called donor-quenching and sensitized emission of the acceptor. Donor-quenching is a reduction of the number of emitted donor photons, while sensitized emission is an increase in emitted acceptor photons. Many microscopic FRET analyses use fluorescence intensity measurements, including acceptor photobleaching2, donor photobleaching2, or FRET-sensitized photobleaching of the acceptor3.
Here, a step-by-step experimental protocol and mathematical algorithm are presented to quantify FRET using donor quenching and acceptor sensitized emission4,5, a method often referred to as ratiometric FRET. Many protocols on how to approximate sensitized emission have been published, few have quantified the absolute FRET efficiency6,7,8,9. The quantification of FRET efficiencies in the living cell requires determining (i) the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins and, also (ii) the detection efficiency of the microscopic setup. While crosstalk can be assessed by imaging cells expressing only one of the fluorophores, the assessment of the relative detection efficiency of the donor and acceptor fluorescence is more complicated. It requires the knowledge of at least the ratio of the number of donor and acceptor molecules giving rise to the measured signals. The number of fluorophores expressed in live cells varies, however, from cell to cell and is unknown. The so-called &#945;&#160;factor characterizes the relative signal strengths from a single excited donor and acceptor molecule. Knowledge of the factor is a prerequisite for quantitative ratiometric FRET measurements in samples with variable acceptor-to-donor molecule ratios as encountered during live-cell imaging with fluorescent proteins. Using a 1-to-1 donor-acceptor fusion protein as a calibration probe permits the determination of the &#945;&#160;factor and also serves as a positive control. This genetically coupled probe is expressed by cells in unknown total amounts but in a fixed and known relative amount of one-to-one. The following protocol lays out how to construct the 1-to-1 probe and how to use it for quantification of FRET efficiency. A spreadsheet that includes all formulae can be found in the supplement and can be used by the readers to enter their own measurements in the respective columns as outlined below.
While the protocol uses the GFP-Cherry donor/ acceptor pair, the presented approach can be performed with any other FRET pair. The Supplementary File 1 provides details on cyan-yellow pairs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5% Trypsin-EDTA without phenol red (10x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Clontech mCherry N1 vector</td>
			<td>Addgene</td>
			<td>3553</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM without phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>11054020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fugene 6</td>
			<td>Promega</td>
			<td>E2691</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>LabTek 8-well chambers #1.0</td>
			<td>Thermo Fisher Scientific</td>
			<td>12565470</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing protein interactions in live-cells with fret-sensitized emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

Figure 1&#160;shows the images obtained in the donor channel, channel 1 (488, 505-530 nm), the transfer channel, channel 2 (488, &#62;585 nm), and the acceptor channel, channel 3 (561, &#62;585 nm), respectively. Representative images of cells expressing GFP only, Cherry only, co-expressing GFP and Cherry, and expressing the GFP-Cherry fusion protein. The mean cellular FRET efficiencies calculated in NRK cells expressing GFP-Cherry fusion protein (positive co...

Watch this Scientific Journal Video about Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission at JoVE.com

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...

This protocol describes a step-by-step experimental process and mathematical algorithm to quantify FRET using donor quenching and acceptor sensitized emission. The quantification of FRET efficiencies in the living cell requires determining the crosstalk of the fluorescent proteins and the relative emission and detection efficiencies of the fluorophores in the microscopic setup. Crosstalk can be assessed by imaging cells expressing only one fluorophore.Assessment of the detection efficiency of a donor or an acceptor molecule requires a knowledge of the ratio of the number of donor and accepter molecules giving rise to the measure signal. The number of fluorophores expressed in live cells varies from cell to cell and is unknown. The calibration probe used in this method, a one-to-one donor-acceptor fusion protein, makes it possible to determine the relative detection efficiency of donor and acceptor molecules.To begin, use an N1 mammalian cell expression vector with mCherry 1 for generating the EGFP mCherry 1 fusion probe. Design oligonucleotides to amplify EGFP without a stop codon as a cell one BAM h1 fragment. The five amino acid linker between the green and red fluorescent protein should yield a mean FRET deficiency for the GFP cherry donor-acceptor pair of 0.25 to 0.3.Use any cell line and media without phenol red to reduce background fluorescence. Once cells are 80%confluent, detach them with one milliliter of 0.05%trypsin EDTA and seed about 10, 000 cells into each well of an eight-well plate by adding three drops from a five milliliter cell suspension. 24 hours after plating, transfect the cells using an appropriate transfection media.Incubate the cells for 20 hours before live cell imaging to allow for proper fluorescent protein expression, folding and maturation. Image the cells in a humidified and heated environmental chamber at 37 degrees Celsius with a confocal scanning microscope. To buffer the cell media at physiological pH, add 20 millimolar HEPES to render the cell media carbon dioxide independent.Then mount the slide on the microscope. Use the 488 nanometer line of the argon ion laser to excite the GFP and the 561 nanometer diode laser to excite cherry. Set up the 488 nanometer laser for excitation in channel one through an emission band of 505 to 530 nanometers and in channel two with a long pass filter of over 585 nanometers.Use the 561 nanometer laser for excitation in channel three with a long pass filter of over 585 nanometers. Excite the two lasers sequentially and set the imaging mode to switch after each line so that the excitation of the 512 by 512 pixel image alternates after each line. Set up a mini time series of three images to detect if significant photobleaching occurs and reduce the laser power if necessary.First, image cells expressing the GFP cherry fusion construct. Set the parameters that define time-integrated laser intensity per pixel in a confocal image. Use a 63X oil objective and zoom set to 3X to image a cell in its entirety with sufficient magnification and resolution.Aim for a pixel size of 70 to 80 nanometers. Set pixel dwell time to two to four microseconds and AOTF transmission for the 488 nanometer and 561 nanometer laser such that images show a good signal-to-noise ratio without any bleaching and no pixels indicating fluorescence intensity saturation. Adjust the laser power of 488 and 561 such that signal levels in channel one and channel three are similar.Set the photo multiplier gain to 600 to 800. Image 15 to 20 cells expressing the GFP cherry fusion protein, cells expressing GFP, cherry, GFP and cherry, and non-transected cells. Then image cells co-expressing the proteins of interest coupled to GFP and cherry respectively.To calculate FRET, measure the donor signal in channel one, the donor channel with 488 nanometer excitation and an emission band of 505 to 530 nanometers. Then measure the acceptor signal in channel three, the acceptor channel with 561 nanometer excitation and emission at above 585 nanometers. Finally, measure the FRET signal in channel two, the transfer channel with 488 nanometer excitation and emission at above 585 nanometers.The signal in channel two is a sum of four different components:the spectral spillover from the quenched donor signal into the over 585 detection channel with a crosstalk factor S1, the acceptor signal from direct excitation by 488 nanometer light with a crosstalk factor S2, the sensitized emission of the acceptor by FRET from the excited donor molecule, and the background signal. Images were obtained in the donor channel, the transfer channel, and the acceptor channel, cells expressing GFP only, cherry only, co-expressing GFP and cherry, and the GFP cherry fusion protein are shown. The mean cellular FRET efficiencies calculated in NRK cells expressing GFP cherry fusion protein and those co-expressing GFP cherry are plotted versus the acceptor-to-donor ratio intensity ratio or molecular ratio in each cell.Normalized pixel by pixel FRET images of cells expressing the GFP cherry fusion protein, co-expressing GFP and cherry as a negative control, and expressing receptor subunits of the Ashwell-Morell receptor are shown here. RHL1 and 2 of the rat hepatic lectin were labeled with GFP and cherry on the cytoplasmic side of the plasma membrane. The mean cellular FRET efficiencies of cells expressing GFP RHL1 and cherry RHL2 and GFP RHL1 delta stock and cherry RHL2 are plotted versus the acceptor-to-donor molecular ratio.The presented quantitative FRET approach allows for the following. Detection of protein interactions in the physiological context of the living cell. Changes in protein interactions over time.Differences in interactions in subcellular compartments down to the pixel by pixel level of a confocal image. And the dependence of the detected FRET signal upon the molecular acceptor-to-donor ratio expressed in a live cell.

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2.9K Görüntüleme.  University of Debrecen. Bu protokol, donör söndürme ve alıcı duyarlılaştırılmış emisyon kullanarak FRET'yi ölçmek için adım adım deneysel bir süreç ve matematiksel algoritmayı açıklar. Canlı hücredeki FRET verimliliklerinin miktarının belirlenmesi, floresan proteinlerinin çapraz etkileşiminin ve mikroskobik kurulumdaki floroforların göreceli emisyon ve algılama verimliliklerinin belirlenmesini gerektirir. Crosstalk, sadece bir florofor eksprese eden görüntüleme hücreleri ile değerl...