1. Cell culture
<ol>
	<li>Maintain cells in a humidified 37 &#176;C/5% CO2 incubator and culture with either DMEM supplemented with 10% fetal bovine serum (FBS) (HeLa and A431 cells) or RPMI supplemented with 15% FBS (U266B1 cells). Culture the cells until they reach 70-80% confluence, then trypsinize them and passage or use them for the assays. 
	&#8203;NOTE: Culture media contained phenol red. No serum starvation was conducted for any cell line prior to conducting the assays.</li>
</ol>
2. Stimulator or inhibitor titration using the two-plate assay protocol with adherent cells
NOTE: This procedure describes how to determine stimulator or inhibitor potencies by generating a concentration-response curve from a dilution series of the test compound.
<ol>
	<li>Cell seeding
	<ol>
		<li>Dispense 50 &#181;L of cells at the pre-optimized density (40,000 HeLa cells/well for both STAT3 and STAT6; 75,000 A431 cells/well for STAT5) into a 96-well tissue culture-treated plate in the appropriate culture medium. Incubate overnight at 37 &#176;C/5% CO2. 
		&#8203;NOTE: Optimal cell density and culture incubation time need to be determined.</li>
	</ol>
	</li>
	<li>Dilutions of test compounds
	<ol>
		<li>Prepare intermediate 2x dilution series of test compound(s) by serially diluting compound(s) (half-log interval dilutions) across 12 wells of a polypropylene 96-well plate into serum-free medium. 
		NOTE: It is recommended to conduct a 12-point, half-log interval concentration-response curve in at least duplicate for an accurate estimation of the EC50 or IC50.</li>
		<li>Alternatively, for hydrophobic, dimethylsulfoxide (DMSO)-soluble test compounds, perform the initial dilutions in 100% DMSO, and then dilute the compound dilution series into serum-free medium. 
		&#8203;NOTE: The assay tolerance to DMSO must be established before conducting a test compound titration in DMSO vehicle. It is important to keep equal solvent concentrations between treated and untreated cells. In addition, when testing serial dilutions of compounds, the solvent concentrations should always remain constant across the dilution series.</li>
	</ol>
	</li>
	<li>Cell treatment
	<ol>
		<li>For cell stimulation, add 50 &#181;L of serum-free medium alone (untreated cells) or containing the stimulator (2x).</li>
		<li>Incubate for the pre-optimized time at either room temperature (RT) or 37 &#176;C (interferon (IFN) &#945;2b/20 min at RT for STAT3; epidermal growth factor (EGF)/10 min at RT for STAT5; interleukin (IL)-4/20 min at RT for STAT6). Proceed then to section 2.4. 
		NOTE: Optimal incubation temperature needs to be determined.</li>
		<li>For cell inhibition, add 25 &#181;L of serum-free medium alone (untreated cells) or containing the inhibitor (4x).</li>
		<li>Incubate for the pre-optimized time at either RT or 37 &#176;C (JAK Inhibitor 1/30 min at RT for STAT3 and STAT6; Erlotinib/15 min at RT for STAT5).</li>
		<li>Add 25 &#181;L of serum-free medium alone (untreated cells) or containing the stimulator (4x) at its EC80.</li>
		<li>Incubate for the pre-optimized time at either RT or 37 &#176;C (same conditions as for step 2.3.2).</li>
	</ol>
	</li>
	<li>Cell lysis
	<ol>
		<li>Prepare the kit&#39;s specific 1x Supplemented Lysis Buffer as indicated by the manufacturer. 
		&#8203;NOTE: It is mandatory to supplement the 1x Lysis Buffer with the 100x Phosphatase Inhibitor Cocktail diluted to a final concentration of 1x. The 1x Supplemented Lysis Buffer contains 1 mM sodium fluoride, 2 mM sodium orthovanadate, and 2 mM beta-glycerophosphate. Other phosphatase inhibitors are not required and should be avoided. Lysis buffers and phosphatase inhibitors other than those included in the kit are not recommended as they might contain ingredients that could interfere with the measurement.</li>
		<li>Carefully remove and discard the cell culture medium by aspirating the supernatant.</li>
		<li>Immediately add 50 &#181;L of 1x Supplemented Lysis Buffer. 
		NOTE: Lysis Buffer volume (25-50 &#181;L) may be optimized.</li>
		<li>Incubate for 30 min at RT under shaking (orbital plate shaker set at 400 rpm; moderate agitation). 
		&#8203;NOTE: Lysis incubation time (30-60 min) may be optimized. Lysates can be used immediately for target protein detection or frozen at -80 &#176;C.</li>
	</ol>
	</li>
	<li>TR-FRET detection
	<ol>
		<li>Prepare the 4x Antibody Detection Mix in 1x Detection Buffer as indicated by the manufacturer.</li>
		<li>In this transfer step, carefully pipette 15 &#181;L of cell lysate from the 96-well culture plate to a well of a white, low-volume 384-well microplate.</li>
		<li>Add 15 &#181;L of the Positive Control Lysate and 15 &#181;L of 1x Lysis Buffer (negative control) to separate assay wells.</li>
		<li>Add 5 &#956;L of 4x Antibody Detection Mix (either Eu-Ab1/FR-Ab2 for detection of the phospho-protein or Eu-Ab3/FR-Ab4 for detection of the total protein) to each of the assay wells.</li>
		<li>Cover the plate with a plate sealer and incubate for 1 h up to overnight at RT, depending on the assay (see the corresponding Technical Data Sheet). 
		NOTE: Optimal reading time needs to be optimized for each assay and cell line. The plate can be read several times without a negative effect on the assay performance.</li>
		<li>Remove the adhesive plate sealer and read the plate on a TR-FRET compatible microplate reader. 
		&#8203;NOTE: Filter-based fluorometers are recommended, though some monochromator instruments can be used. Verify that the appropriate optic module (filters and mirror) for TR-FRET is installed. Use an excitation wavelength of 320 or 340 nm to excite the Europium chelate. Read assays at both 615 nm (or 620 nm) and 665 nm to detect both the emission from the donor Europium and the acceptor fluorophore, respectively. The instrument settings will depend on the particular reader. Data presented here were obtained using lamp-based excitation, 90 &#181;s delay, 300 &#181;s integration time, and 100 flashes per well. The phospho-STAT4 assay, however, was read using laser excitation to generate higher signal-to-background (S/B) ratios.</li>
	</ol>
	</li>
</ol>
3. Stimulator or inhibitor titration using the two-plate assay protocol with suspension cells
<ol>
	<li>Dilution of test compounds
	<ol>
		<li>Prepare intermediate 2x dilution series of test compound(s) as described in steps 2.2.1 and 2.2.2.</li>
	</ol>
	</li>
	<li>Cell seeding and treatment
	<ol>
		<li>Dispense 20 &#181;L of cells at the pre-optimized density (200,000 U266B1 cells/well for STAT1; 400,000 U266B1 cells/well for STAT4) into a 96-well tissue culture-treated plate in the appropriate culture medium. Directly proceed to cell treatment or incubate 2-4 h at 37 &#176;C, 5% CO2. 
		NOTE: This step needs to be optimized for different cell types.</li>
		<li>For cell stimulation, add 20 &#181;L of serum-free medium alone (untreated cells) or containing the stimulator (2x).</li>
		<li>Incubate for the pre-optimized time at either RT or 37 &#176;C (IFN&#945;2b/15 min at RT for STAT1; IFN&#945;2b/25 min at 37 &#176;C for STAT4). Proceed then to section 3.3. 
		NOTE: Optimal incubation temperature needs to be determined.</li>
		<li>For cell inhibition, add 10 &#181;L of serum-free medium alone (untreated cells) or containing the inhibitor (4x).</li>
		<li>Incubate for the pre-optimized time at either RT or 37&#160;&#176;C (JAK Inhibitor 1/30 min at RT for STAT1 and STAT4).</li>
		<li>Add 10 &#181;L of serum-free medium alone (untreated cells) or containing the stimulator (4x) at its EC80.</li>
		<li>Incubate for the pre-optimized time at either RT or 37&#160;&#176;C (same conditions as for step 3.2.3).</li>
	</ol>
	</li>
	<li>Cell lysis
	<ol>
		<li>Prepare the kit&#39;s specific 5x Supplemented Lysis Buffer as indicated by the manufacturer. 
		NOTE: It is mandatory to supplement the 5x Lysis Buffer with the 100x Phosphatase Inhibitor Cocktail diluted to a final concentration of 5x. The 5x Supplemented Lysis Buffer contains 5 mM sodium fluoride, 10 mM sodium orthovanadate, and 10 mM beta-glycerophosphate. Other phosphatase inhibitors are not required and should be avoided.</li>
		<li>Add 10 &#181;L of 5x Supplemented Lysis Buffer.</li>
		<li>Incubate for 30 min at RT under shaking (orbital plate shaker set at 400 rpm; moderate agitation). 
		&#8203;NOTE: Lysis incubation time (30-60 min) may be optimized. Lysates can be used immediately for target protein detection or frozen at -80&#160;&#176;C.</li>
	</ol>
	</li>
	<li>TR-FRET detection
	<ol>
		<li>Following cell lysis, conduct the TR-FRET detection step as described in section 2.5 for the 2-plate assay protocol for adherent cells.</li>
	</ol>
	</li>
</ol>
4. Stimulator titration using the one-plate assay protocol with adherent or suspension cells
<ol>
	<li>Dilution of test compounds
	<ol>
		<li>Prepare intermediate dilution series of test compound(s) at 3x by serially diluting compound(s) (half-log interval dilutions) across 12 wells of a polypropylene 96-well plate into serum-free medium.</li>
	</ol>
	</li>
	<li>Cell seeding and treatment
	<ol>
		<li>Dispense 8 &#181;L of cells at the pre-optimized density (160,000 U266B1 cells/well for STAT4; 80,000 HeLa cells/well for STAT6), in the appropriate serum-free culture medium, into a white, low-volume 384well assay plate. Directly proceed to cell treatment or incubate 2-4 h at 37&#160;&#176;C, 5% CO2. 
		NOTE: The requirement for a cell culture incubation period before treatment needs to be determined for different cell types.</li>
		<li>For cell stimulation, add 4 &#181;L of serum-free medium alone (untreated cells) or containing the stimulator (3x).</li>
		<li>Incubate for the pre-optimized time at either room temperature or 37&#160;&#176;C (IFN&#945;2b/25 min at 37&#176;C for STAT4; IL-4/20 min at 37&#176;C for STAT6). Proceed then to section 4.3.</li>
	</ol>
	</li>
	<li>Cell lysis
	<ol>
		<li>Prepare the kit&#39;s specific 5x Supplemented Lysis Buffer as indicated by the manufacturer. 
		NOTE: It is mandatory to supplement the 5x Lysis Buffer with the 100x Phosphatase Inhibitor Cocktail diluted to a final concentration of 5x.</li>
		<li>Add 3 &#181;L of 5x Supplemented Lysis Buffer.</li>
		<li>Incubate for 30 min at RT under shaking (orbital plate shaker at 400 rpm). 
		NOTE: Lysis incubation time (30-60 min) may be optimized. Lysates can be used immediately or frozen at 80 &#176;C.</li>
	</ol>
	</li>
	<li>TR-FRET detection
	<ol>
		<li>Add 15 &#181;L of Positive Control Lysate (undiluted) and 15 &#181;L of 1x Supplemented Lysis Buffer (negative control) to separate assay wells.</li>
		<li>Add 5 &#181;L of 4x Antibody Detection Mix (either Eu-Ab1/FR-Ab2 for the phospho-protein or Eu-Ab3-/FR-Ab4 for the total protein) prepared in 1x Detection Buffer to each of the assay wells.</li>
		<li>Cover the plate with a plate sealer and incubate for 1 h up to overnight at RT, depending on the assay. 
		NOTE: Optimal reading time needs to be optimized for each assay and cell line. The plate can be read several times without a negative effect on the assay performance.</li>
		<li>Remove the adhesive plate sealer. Read the plate on a TR-FRET-compatible microplate reader.</li>
	</ol>
	</li>
</ol>
5. Data analysis
<ol>
	<li>Calculate the TR-FRET ratio for each well using the following formula (1): 
	<img alt="Equation 1" src="/files/ftp_upload/62915/62915eq01.jpg" /> (1) 
	NOTE: Because the TR-FRET signal is read in a time-resolved mode, background subtraction is usually not necessary. If background subtraction is conducted, use the cell-free wells containing the 1x Supplemented Lysis Buffer (negative control) for background subtraction. Determine the average TR-FRET ratio from the cell-free wells and then subtract this value from the TR-FRET ratio of each well.</li>
	<li>For concentration-response curves, analyze the data according to a nonlinear regression using the 4 parameter logistic equation (sigmoidal dose-response curve with variable slope) and a 1/Y2 data weighting to generate EC50 or IC50 values.</li>
	<li>For the Z&#39; factor experiment, analyze the data according to the following formula (2)10 
	<img alt="Equation 2" src="/files/ftp_upload/62915/62915eq02.jpg" /> (2) 
	Where &#181; and &#963; are the mean values and standard deviations for the positive control (p; stimulated cells) and negative control (n; untreated cells), respectively.</li>
</ol>

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+JAK-STAT+pathway:+impact+on+human+disease+and+therapeutic+intervention.">The JAK-STAT pathway: impact on human disease and therapeutic intervention.</a> Annual Review of Medicine. 66, 311-328 (2015).</li><li>Verhoeven, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.+potential+and+controversy+of+targeting+STAT+family+members+in+cancer.">et al. potential and controversy of targeting STAT family members in cancer.</a> Seminars in Cancer Biology. 60 (2), 41-56 (2020).</li><li>Gilda, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.+blotting+inaccuracies+with+unverified+antibodies:+need+for+a+Western+blotting+minimal+reporting+standard+(WBMRS).">et al. blotting inaccuracies with unverified antibodies: need for a Western blotting minimal reporting standard (WBMRS).</a> PLoS One. 10 (8), 0135392 (2015).</li><li>Binder, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.+and+utilization+of+the+SureFire+phospho-STAT5+assay+for+a+cell-based+screening+campaign.">et al. and utilization of the SureFire phospho-STAT5 assay for a cell-based screening campaign.</a> Assay and Drug Development Technologies. 6 (1), 27-37 (2008).</li><li>Ayoub, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogeneous+time-resolved+fluorescence-based+assay+to+monitor+extracellular+signal-regulated+kinase+signaling+in+a+high-throughput+format.">Homogeneous time-resolved fluorescence-based assay to monitor extracellular signal-regulated kinase signaling in a high-throughput format.</a> Frontiers in Endocrinology. 5, 94 (2014).</li><li>Robers, M. B., Machleidt, T., Carlson, C. B., Bi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+LanthaScreen+and+beta-lactamase+reporter+assays+for+high-throughput+screening+of+JAK2+inhibitors.">Cellular LanthaScreen and beta-lactamase reporter assays for high-throughput screening of JAK2 inhibitors.</a> Assay and Drug Development Technologies. 6 (4), 519-529 (2008).</li><li>Hwang, B., Engel, L., Goueli, S. 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Competing Interests: Jaime Padros, Mireille Caron, and Genevi&#232;ve Chatel are employees of BioAuxilium Research, which manufactures the THUNDER TR-FRET assay kits used in this study. In addition, Jaime Padros and Mireille Caron are shareholders of BioAuxilium Research. This does not alter the authors&#39; adherence to all JoVE policies on sharing data and materials.

Compared to conventional methods for phosphoprotein analysis such as western blotting and ELISA-based methods, the workflow for a THUNDER TR-FRET cellular assay is simple and fast, uses a low-volume sample (15 &#181;L), is designed for HTS in a 384-well format, and is highly amenable to automation. The assay protocol is flexible and can readily be adapted to both medium- and high-throughput applications. Assays can be run using either a two-plate transfer protocol or a one-384-well plate protocol. In the two-plate transf...

The JAK/STAT signaling pathway plays a key role in mediating cellular responses to diverse cytokines, interferons, growth factors, and related molecules1,2. The binding of these ligands to specific cell-surface receptors results in the activation of JAKs, which in turn activate STAT proteins by phosphorylation of specific tyrosine residues. STAT phosphorylation results in their dimerization and translocation into the nucleus, where they exert their effect on the transcription of regulated target genes. The STAT family consists of seven members: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. The members play a complex and essential role in the regulation of physiologic cell processes, including proliferation, differentiation, apoptosis, angiogenesis, and immune system regulation. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions3,4. Therefore, the ability to assess STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research.
To date, the conventional methods used to measure intracellular phosphorylated protein levels, including STATs, are antibody-based and include western blotting, ELISA, and phosphoflow cytometry. These heterogeneous methods are labor-intensive, time-consuming, error-prone, low-throughput, and often unreliable (e.g., specificity issues) in the case of western blotting5. In contrast, homogeneous assays require fewer experimental steps, use smaller sample volumes, and are amenable to HTS. There are five homogeneous cell-based immunoassay platforms commercially available that can be used to quantitatively monitor JAK-dependent phosphorylation of STATs in cell lysates: SureFire, HTRF, LANCE, LanthaScreen, and Lumit. Each of these platforms has its advantages and disadvantages.
SureFire is based on luminescent oxygen channeling technology, which utilizes donor and acceptor beads coated to specifically capture a pair of antibodies, one of which is biotinylated. In the presence of phosphorylated protein, the two antibodies bring the donor and acceptor beads into close proximity, enabling the generation of a chemiluminescent signal6. While versatile and sensitive, this technology is expensive, is affected by biotin in the culture medium, is very sensitive to ambient temperature and light, and requires a special reader for detection. HTRF and LANCE are both based on TR-FRET technology that utilizes long-lifetime luminescent lanthanide ion complexes (Europium or Terbium chelates, or Europium cryptate) as the donor molecules and far-red fluorophores as the acceptor molecules7. When two protein-specific antibodies labeled with either donor or acceptor molecules are brought into close proximity, FRET takes place, causing an increase in acceptor fluorescence and a decrease in donor fluorescence. These long-lived fluorescent signals can be measured in a time-resolved and ratiometric manner to reduce assay interference and increase data quality. Other advantages of TR-FRET are that it is not light-sensitive, allows repeated readings, and exhibits long signal stability. While TR-FRET is widely implemented in HTS due to its versatility, sensitivity, and high robustness, all commercial TR-FRET-based assay platforms are expensive, thereby precluding its wide adoption in academic and small industrial laboratories. The LanthaScreen assay also uses a TR-FRET based-readout but is reliant on an engineered U2OS cell line that stably expresses green fluorescent protein (GFP)-STAT1 fusion protein combined with a terbium-labeled phospho-specific STAT1 antibody8. In addition to being limited in terms of choice of signaling proteins, this method requires purchasing expensive transfected cell lines, reducing its applicability and increasing the possibility of experimental artifacts. Lumit is a generic bioluminescent immunoassay platform that utilizes secondary antibodies (anti-mouse and anti-rabbit) chemically labeled with the small and large NanoBit subunits of NanoLuc Luciferase9. The binding of two primary antibodies to the target protein brings the secondary antibodies into proximity to form an active enzyme that generates a luminescence signal. While luminescence is generally a sensitive and robust readout, the requirement for primary antibodies raised in two different species limits the choices for assay design. In addition, the use of secondary antibodies in complex sample matrixes may be prone to assay interference.
Thus, a need still exists for a reliable, rapid, yet affordable cell-based assay platform for measuring individual phosphorylated and total STAT proteins in a manner compatible with HTS. To address this need, a new high-throughput cell-based immunoassay platform was developed based on an enhanced TR-FRET technology (THUNDER) and designed to enable simple, sensitive, robust, and cost-effective measurement of endogenously expressed intracellular proteins (phosphorylated or total) in cell lysates. The advantages of this technology stem from the combination of a donor/acceptor FRET pair exhibiting exceptional spectral compatibility and TR-FRET signal, rigorously validated antibodies, and optimized lysis buffers. These assays are formatted as sandwich immunoassays and use a straightforward, three-step workflow (Figure 1). Cells are first treated to modulate protein phosphorylation and then lysed with the specific lysis buffer provided in the kit. The target phosphorylated or total STAT protein in the cell lysate is detected in a single reagent addition and incubation step with a pair of fluorophore-labeled antibodies that recognize distinct epitopes on the target protein (Figure 2). One antibody is labeled with a Europium chelate donor (Eu-Ab1), while the second antibody is labeled with a far-red acceptor fluorophore (FR-Ab2). The two labeled antibodies bind to the protein in solution, bringing the two labels into close proximity. Excitation of the donor Europium chelate at 320 or 340 nm triggers a FRET to the acceptor, which emits a long-lived TR-FRET signal at 665 nm proportional to the concentration of target protein (phosphorylated or total) in the cell lysate.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62915/62915fig01.jpg" /> 
Figure 1: TR-FRET assay workflow. The workflow consists of three steps: cell treatment, cell lysis, and protein detection using TR-FRET. In the two-plate assay protocol, lysates are transferred to a white 384-well detection plate, whereas in the one-plate protocol, all steps are conducted in the same white 384-well detection plate (all-in-one-well protocol). Regardless of the assay protocol used, protein detection is performed in the same total volume (20 &#181;L per well). Abbreviation: TR-FRET = time-resolved F&#246;rster resonance energy transfer. <a href="https://www.jove.com/files/ftp_upload/62915/62915fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62915/62915fig02.jpg" /> 
Figure 2: TR-FRET sandwich immunoassay principle. One antibody is labeled with the Europium chelate donor (Eu-Ab1) and the second with the far-red small fluorophore acceptor (FR-Ab2). The two labeled antibodies bind specifically to distinct epitopes on the target protein (phosphorylated or total) in the cell lysate, bringing the two fluorophores into close proximity. Excitation of the donor Europium chelate at 320 or 340 nm triggers a FRET from the donor to the acceptor molecules, which in turn emit a signal at 665 nm. This signal is proportional to the concentration of protein in the cell lysate. In the absence of the specific target protein, the donor and acceptor fluorophores are too distant from each other for FRET to occur. Abbreviations: FRET = F&#246;rster resonance energy transfer; TR-FRET = time-resolved FRET; Ab = antibody; FR = far-red; Eu - Europium chelate; P = phosphorylation. <a href="https://www.jove.com/files/ftp_upload/62915/62915fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Here, detailed protocols are provided for measuring, in a 384-well format, the intracellular levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641), together with total STAT1, STAT3, STAT5, and STAT6, in cell lysates from adherent or suspension cells using the THUNDER TR-FRET platform. These protocols define steps for cell treatment, lysis, and TR-FRET-based target protein detection using either a two-plate transfer protocol or a one-plate all-in-one-well protocol. These cell-based assays are applied for determining the pharmacological profile of known activators and inhibitors of the JAK/STAT pathway. The robustness and suitability of selected assays for HTS are demonstrated. Lastly, key experiments for assay optimization are discussed, along with recommendations for assay troubleshooting.

<table><tbody><tr><td>96-well microplate, clear, flat bottom, polystyrene, tissue culture-treated, sterile</td><td>Corning</td><td>3595</td><td>This is the plate for culturing cells when using the two-plate assay protocol. Other cell culture 96-well plates can be used</td></tr><tr><td>384-well microplate, white, low-volume</td><td>PerkinElmer</td><td>6007290</td><td>This is the plate for TR-FRET detection when using the two- or one-plate assay protocols. Other low-volume, white 384-well plates can be used</td></tr><tr><td>A431 cell line</td><td>ATCC</td><td>CRL-1555</td><td></td></tr><tr><td>Adhesive microplate seal</td><td>PerkinElmer</td><td>6050185</td><td></td></tr><tr><td>DMSO</td><td>Fisher</td><td>D159-4</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium (DMEM)</td><td>Wisent</td><td>319-005-CL&amp;nbsp;</td><td>THUNDER TR-FRET is compatible with culture medium containing phenol red</td></tr><tr><td>EnVision Xcite Multilabel plate reader</td><td>PerkinElmer</td><td>2104-0020A</td><td>The assays can be performed on a variety of plate readers equipped with the TR-FRET option</td></tr><tr><td>Erlotinib hydrochloride</td><td>Sigma</td><td>CDS022564</td><td></td></tr><tr><td>Falcon tissue culture treated flasks</td><td>Fisher</td><td>13-680-65</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Wisent</td><td>098-150</td><td></td></tr><tr><td>HeLa cell line</td><td>ATCC</td><td>CCL-2</td><td></td></tr><tr><td>JAK Inhibitor 1</td><td>Cayman Chemical</td><td>15146</td><td></td></tr><tr><td>Orbital plate shaker</td><td>Many options available</td><td>Not applicable</td><td></td></tr><tr><td>Recombinant human EGF</td><td>PeproTech</td><td>AF-100-15</td><td></td></tr><tr><td>Recombinant human IFN&amp;alpha;2b</td><td>ProSpec</td><td>CYT-460</td><td></td></tr><tr><td>Recombinant human IL-4</td><td>R&amp;amp;D Systems</td><td>204-IL</td><td></td></tr><tr><td>Roswell Park Memorial Institute 1640 medium (RPMI)</td><td>Wisent</td><td>350-007-CL</td><td>THUNDER TR-FRET is compatible with culture medium containing phenol red</td></tr><tr><td>THUNDER Phospho-STAT1 (Y701) + Total STAT1 TR-FRET Cell Signaling Assay Kit</td><td>BioAuxilium Research</td><td>KIT-STAT1PT-500</td><td></td></tr><tr><td>THUNDER Phospho-STAT3 (Y705) + Total STAT3 Cell Signaling Assay Kit</td><td>BioAuxilium Research</td><td>KIT-STAT3PT-500</td><td></td></tr><tr><td>THUNDER Phospho-STAT4 (Y693) TR-FRET Cell Signaling Assay Kit</td><td>BioAuxilium Research</td><td>KIT-STAT4P-500</td><td></td></tr><tr><td>THUNDER Phospho-STAT5 (Y694/Y699) + Total STAT5 TR-FRET Cell Signaling Assay Kit</td><td>BioAuxilium Research</td><td>KIT-STAT5PT-500</td><td></td></tr><tr><td>THUNDER Phospho-STAT6 (Y641) + Total STAT6 TR-FRET Cell Signaling Assay Kit</td><td>BioAuxilium Research</td><td>KIT-STAT6PT-500</td><td></td></tr><tr><td>Trypsin/EDTA 0.05%</td><td>Wisent</td><td>325-542-CL</td><td></td></tr><tr><td>U266B1 cell line</td><td>ATCC</td><td>TIB-196</td><td></td></tr><tr><td>Ultrapure water</td><td>NA</td><td>NA</td><td>Use Milli-Q grade water (18 M&amp;Omega;.cm) to dilute Lysis Buffer and Detection Buffer</td></tr></tbody></table>

time-resolved f&#246;rster resonance energy transfer assays for measurement of endogenous phosphorylated stat proteins in human cells

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to cytokines and growth factors. STAT proteins are activated by tyrosine phosphorylation mediated mainly by JAKs. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions. Therefore, the ability to monitor STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research. The traditional assay formats available to quantify phosphorylated STAT proteins include western blotting and the enzyme-linked immunosorbent assay (ELISA). These heterogeneous methods are labor-intensive, low-throughput, and often not reliable (specific) in the case of western blotting. Homogeneous (no-wash) methods are available but remain expensive. 
Here, detailed protocols are provided for the sensitive, robust, and cost-effective measurement in a 384-well format of endogenous levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641) in cell lysates from adherent or suspension cells using the novel THUNDER time-resolved F&#246;rster resonance energy transfer (TR-FRET) platform. The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). The assay protocol is flexible, uses a low-volume sample (15 &#181;L), requires only one reagent addition step, and can be adapted to low-throughput and high-throughput applications. Each phospho-STAT sandwich immunoassay is validated under optimized conditions with known agonists and inhibitors and generates the expected pharmacology and Z'-factor values. As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches. Together, this suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.

Each THUNDER TR-FRET assay was pharmacologically validated by treating adherent (HeLa or A431) or suspension cells (U266B1) with JAK/STAT pathway-specific activators or inhibitors and then measuring the levels of specific phosphorylated and total STATs, when applicable. Assays were conducted in 384-well format using the two-plate transfer protocol and pre-optimized assay conditions. Figure 3, Figure 4, Figure 5, <strong class="xfig"...

Watch this Scientific Journal Video about Time-resolved FÃ¶rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells at JoVE.com

Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

Time-resolved F&#246;rster resonance energy transfer cell-based assay protocols are described for the simple, specific, sensitive, and robust quantification of endogenous phosphorylated signal transducer and activator of transcription (STAT) 1/3/4/5/6 proteins in cell lysates in a 384-well format.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to ...

time-resolved-frster-resonance-energy-transfer-assays-for-measurement

Research

JoVE Journal

Biochemistry

3.4K Views.  BioAuxilium Research. Time-resolved Förster resonance energy transfer cell-based assay protocols are described for the simple, specific, sensitive, and robust quantification of endogenous phosphorylated signal transducer and activator of transcription (STAT) 1/3/4/5/6 proteins in cell lysates in a 384-well format. 

Video: Time-resolved Förster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Bioengineering

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser scanning microscopes have become a workhorse for biologists. Commercial systems offer high flexibility in laser power adjustment and detector sensitivity and often combine different detectors to obtain the perfect image. However, the comparison of intensity-based data from different experiments and setups is often impossible due to this flexibility. Biologist-friendly procedures are of advantage and allow for simple and reliable adjustment of laser and detector settings. 
Furthermore, as FRET experiments in living cells are affected by the variability in protein expression and donor-acceptor ratios, protein expression levels must be considered for data evaluation. Described here is a simple protocol for reliable and reproducible FRET measurements, including routines for the estimation of protein expression and adjustment of laser intensity and detector settings. Data evaluation will be performed by calibration with a fluorophore fusion of known FRET efficiency. To improve simplicity, correction factors have been compared that have been obtained in cells and by measuring recombinant fluorescent proteins.

A protocol is provided for setting up a standard confocal laser-scanning microscope for in vivo F&#246;rster resonance energy transfer measurements, followed by data evaluation.

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

F&#246;rster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between biomolecules in vitro as well as within cells. In FRET, the efficiency of energy transfer, measured by changes in fluorescence intensity or lifetime, relates to the distance between two fluorescent molecules or labels. Determination of dynamics and conformational changes from the distances are just some examples of applications of this method to biological systems. Under certain conditions, this methodology can add to and enhance existing X-ray crystal structures by providing information regarding dynamics, flexibility, and adaptation to binding surfaces. We describe the use of FRET and associated distance determinations to elucidate structural properties, through the identification of a binding site or the orientations of dimer subunits. Through judicious choice of labeling sites, and often employment of multiple labeling strategies, we have successfully applied these mapping methods to determine global structural properties in a protein-DNA complex and the SecA-SecYEG protein translocation system. In the SecA-SecYEG system, we have used FRET mapping methods to identify the preprotein-binding site and determine the local conformation of the bound signal sequence region. This study outlines the steps for performing FRET mapping studies, including identification of appropriate labeling sites, discussion of possible labels including non-native amino acid residues, labeling procedures, how to perform measurements, and interpreting the data.

The study details the methodology of FRET mapping including the selection of labeling sites, choice of dyes, acquisition, and data analysis. This methodology is effective at determining binding sites, conformational changes, and dynamic motions in protein systems and is most useful if performed in conjunction with existing 3-D structural information.

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Biology

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Medicine

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...

Neuroscience

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.

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 ...

Single-Molecule Pull-Down Assay for Protein Phosphorylation Analysis: A High Throughput Technique to Quantify Protein Phosphorylation in Cell Lysate

Source: Bailey, E. M. et al., <a href="https://www.jove.com/v/63665">An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation.</a> J. Vis. Exp. (2022)
This video demonstrates a sensitive quantification technique of protein phosphorylation using a single-molecule pull-down assay. The functionalization of polyethylene glycol-biotin and the use of labeled antibodies increases the detection of phosphorylated tyrosine with specificity.

Source: Bailey, E. M. et al., An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation. J. Vis. Exp. (2022)
This video ...

Time-resolved Förster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

Summary

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Chapters in this video

Quantitative FRET (Förster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Förster Resonance Energy Transfer Measurements in Living Plant Cells

A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

Förster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

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

Single-Molecule Pull-Down Assay for Protein Phosphorylation Analysis: A High Throughput Technique to Quantify Protein Phosphorylation in Cell Lysate