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>
	<li>Villarino, A., Kanno, Y., O'Shea, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+consequences+of+Jak-STAT+signaling+in+the+immune+system.">Mechanisms and consequences of Jak-STAT signaling in the immune system.</a> Nature Immunology. 18 (4), 374-384 (2017).</li><li>Hammar&#233;n, H. M., Virtanen, A. T., Raivola, J., Silvennoinen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+JAKs+in+cytokine+signaling+and+its+breakdown+in+disease.">The regulation of JAKs in cytokine signaling and its breakdown in disease.</a> Cytokine. 118, 48-63 (2019).</li><li>O'Shea, J. 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. A., Zegzouti, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogeneous+bioluminescent+immunoassay+to+probe+cellular+signaling+pathway+regulation.">Homogeneous bioluminescent immunoassay to probe cellular signaling pathway regulation.</a> Communications Biology. 3 (8), 1-12 (2020).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+statistical+parameter+for+use+in+evaluation+and+validation+of+high-throughput+screening+assays.">A simple statistical parameter for use in evaluation and validation of high-throughput screening assays.</a> Journal of Biomolecular Screening. 4 (2), 67-73 (1999).</li><li>Osmond, R. I. W., Das, S., Crouch, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+cell-based+assays+for+cytokine+receptor+signaling,+using+an+AlphaScreen+SureFire+assay+format.">Development of cell-based assays for cytokine receptor signaling, using an AlphaScreen SureFire assay format.</a> Analytical Biochemistry. 403 (1-2), 94-101 (2010).</li><li>Haan, 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=Jak1+has+a+dominant+role+over+Jak3+in+signal+transduction+through+&#947;c-containing+cytokine+receptors.">Jak1 has a dominant role over Jak3 in signal transduction through &#947;c-containing cytokine receptors.</a> Chemistry &amp; Biology. 18 (3), 314-323 (2011).</li><li>Kim, Y., Apetri, M., Luo, B., Settleman, J. E., Anderson, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+tyrosine+kinase+inhibitors+on+normal+and+oncogenic+EGFR+signaling+and+downstream+effectors.">Differential effects of tyrosine kinase inhibitors on normal and oncogenic EGFR signaling and downstream effectors.</a> Molecular Cancer Research. 13 (4), 765-774 (2015).</li><li>Qian, 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=Comparison+of+two+homogeneous+cell-based+kinase+assays+for+JAK2+V617F:+SureFire+pSTAT5+and+GeneBLAzer+fluorescence+resonance+energy+transfer+assays.">Comparison of two homogeneous cell-based kinase assays for JAK2 V617F: SureFire pSTAT5 and GeneBLAzer fluorescence resonance energy transfer assays.</a> ASSAY and Drug Development Technologies. 10 (2), 212-217 (2012).</li><li>Iversen, P. W., et al., Markossian, S., 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=HTS+assay+validation.">HTS assay validation.</a> Assay Guidance Manual. , (2012).</li><li>Muelas, M. W., Ortega, F., Breitling, R., Bendtsen, C., Westerhoff, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+cell+culture+optimization+enhances+experimental+reproducibility+in+cancer+cells.">Rational cell culture optimization enhances experimental reproducibility in cancer cells.</a> Scientific Reports. 8, 3029 (2018).</li></ol>

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 transfer protocol, cells are seeded, treated, and lysed in one cell culture plate, and lysates are subsequently transferred to a separate white detection plate (half-area 96-well or 384-well plate) for analysis. In a one-384-well plate protocol, the entire workflow is conducted in the same well, eliminating the need for a liquid transfer step. The assays could easily be adapted to the 1536-well format by reducing the volume proportionally. Regardless of the assay protocol, there are only three reagent solutions to prepare and no washing steps, and the assays can be run following an &#34;addition-only&#34; protocol, requiring minimal hands-on time.
A critical step in performing any cell-based assay is the optimization of cell culture and treatment conditions16. Both the cell number and treatment conditions require careful optimization before running an assay, as these key parameters often vary for each cell line and each phosphoprotein. The optimization of these parameters allows maximizing the assay window, obtaining an optimum performance with a high S/B ratio and low inter-well coefficient of variation, and ensuring the robustness and reproducibility of the results. Cell number, serum-starvation (when appropriate), and stimulation or inhibition time (at either room temperature or 37&#160;&#176;C) should be optimized for each cell line and target protein. Too high or too low cell numbers can negatively influence the modulation of intracellular signaling pathways. Cell seeding densities of 40,000-80,000 cells/well for adherent cells or 100,000-200,000 cells/well for suspension cells are generally acceptable for most cell lines. Of note, the optimal length of time for stimulation and inhibition can vary widely among cell lines and target proteins, from a few minutes to several hours. As such, a time course study is strongly recommended to determine the optimal stimulation and inhibition incubation times, ideally at both room temperature and 37&#160;&#176;C, as incubation temperature affects the kinetics of target protein stimulation.
Troubleshooting a cell-based assay can be difficult and time-consuming because of the many steps involved in the workflow (cell culture, cell treatment, lysis, and protein detection). These assay kits include a positive control lysate. It is recommended to use these positive control lysates and negative control (1x supplemented Lysis Buffer alone) in every experiment. The use of proper controls facilitates troubleshooting, as problems can then be quickly attributed to either the detection step (incorrect reagent preparation and/or assay execution) or the quality of the lysates used in the experiment. The latter is generally due to the use of suboptimal cellular conditions.
An additional advantage of this platform is the availability of a suite of optimized lysis buffers with different stringencies. This allows generating more or less heterogeneous lysates from partial subcellular fractionation. This is useful because signaling proteins are located in various intracellular compartments, and some, like STAT proteins, shuttle between compartments (e.g., between the cytoplasm and the nucleus). Of note, phosphorylated and total STAT1 use lysis buffer 1 because they cannot be detected well with the lysis buffer 2 used for the other STAT proteins. In contrast, other homogeneous methods rely on a single &#34;universal&#34; lysis buffer, which generates a more complex sample that can create non-specific interactions with the antibodies. However, the use of different lysis buffers may preclude the analysis of multiple signaling proteins from a single lysate sample. Nevertheless, one can still test lysates generated with different lysis buffers simultaneously on a single plate for parallel pathway analysis.
In conclusion, the methods presented here based on this homogeneous TR-FRET cellular immunoassay platform offer a high-throughput alternative for monitoring JAK/STAT signaling via the detection and quantification of endogenous phosphorylated STAT1/3/4/5/6 proteins, together with total STAT1/3/5/6, in cell lysates. These assays provide new tools for a more comprehensive analysis of specific STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathways. Given its simplicity, specificity, sensitivity, reproducibility, and cost-effectiveness, this new assay platform represents an attractive alternative to traditional immunoassays, both in an academic setting and in industrial laboratories for HTS applications.

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#8217;s modified Eagle medium (DMEM)</td>
			<td>Wisent</td>
			<td>319-005-CL&#160;</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&#945;2b</td>
			<td>ProSpec</td>
			<td>CYT-460</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-4</td>
			<td>R&#38;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) + Total STAT4 TR-FRET Cell Signaling Assay Kit</td>
			<td>BioAuxilium Research</td>
			<td>KIT-STAT4PT-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&#937;.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, Figure 6, and Figure 7 summarize representative concentration-response curves obtained for all STAT assays. Overall, all stimulator and inhibitor concentration-response curves for the phospho-STAT assays showed robust TR-FRET signals, wide dynamic ranges, low inter-well coefficient of variation (typically &#8804;5%), and acceptable S/B ratios. The EC50 and IC50 values reported here are within the range of expected values.
Treatment of cells with the JAK activators IFN&#945;2b (phospho-STAT1, phospho-STAT3, and phospho-STAT4), IL-4 (phospho-STAT6), and EGF (phospho-STAT5) showed the anticipated concentration-dependent increase in STAT phosphorylation at specific tyrosine residues, while the corresponding total STAT proteins (STAT1, STAT3, STAT5, and STAT6) remained stable (Figure 3, Figure 4, Figure 5, Figure 6, and Figure 7, Panel A). The signal decrease (less than 20%) observed for total STAT5 with increasing STAT5 phosphorylation is expected and is due to steric hindrance, where phosphorylation of STAT5 hinders the binding of one of the two anti-total STAT5 antibodies to its respective antigen. The EC50 values were in the sub-nanomolar range for IFN&#945;2b and IL-4 (0.083 to 0.47 nM) and in the low nanomolar range for EGF (12 nM). These values are in agreement with published data6,11.
To confirm that the signal induced by the activators was mediated by the activation of endogenous receptors, cells were pretreated with increasing concentrations of either JAK Inhibitor 1 (a pan JAK inhibitor) or Erlotinib (an EGFR tyrosine kinase inhibitor) prior to submaximal stimulation (EC80) with the STAT activators. As anticipated, both JAK Inhibitor 1 and Erlotinib inhibited the corresponding phospho-STAT levels in a concentration-dependent manner, with IC50 values ranging between the low nanomolar and the high nanomolar range for JAK Inhibitor 1 (29-759 nM) and in the low nanomolar range for Erlotinib (10 nM) (Figure 3, Figure 4, Figure 5, Figure 6, and Figure 7, Panel B). The IC50 values are consistent with published data11,12,13. As was the case for stimulation experiments, the levels of corresponding total STAT were not affected by either treatment. Taken together, these results demonstrate the specificity of each assay for its endogenous target STAT protein (phosphorylated or total) and their capacity to profile activators or inhibitors exhibiting a range of potencies.
Assays in which the whole workflow (cell treatment, lysis, and protein detection) is conducted in a single well of a 384-well plate without a transfer step are more suitable for HTS. Accordingly, the phospho-STAT4 and phospho-STAT6 assays were performed using the one-plate protocol. Representative data obtained under optimized conditions are summarized in Figure 8 and Figure 9. Stimulation of either phosphorylated STAT4 by IFN&#945;2b in a suspension cell line (U266B1; Figure 8) or phosphorylated STAT6 by IL-4 in an adherent cell line (HeLa; Figure 9) was obtained with acceptable S/B ratios and EC50 values consistent with those obtained using the two-plate protocol. These data show that the phospho-STAT assays can be successfully adapted from a two-plate transfer protocol to a one-plate all-in-one-well protocol.
The Z&#39;-factor is commonly used in the HTS community for the evaluation of the suitability and robustness of an assay for HTS10. To further validate the phospho-STAT assays, preliminary Z&#39;-factor studies were manually conducted using the two-plate protocol. To assess assay stability, plates were read following both a 4-h incubation and an overnight incubation. Results obtained for the phospho-STAT1 and phospho-STAT3 assays, using a suspension cell line (U266B1) or an adherent cell line (HeLa), respectively, are summarized in Figure 10. Calculated Z&#8242;-factor values were 0.85 for phospho-STAT1 and 0.79 for phospho-STAT3. Following overnight incubation, the Z&#39;-factor values remained stable (0.83 for phospho-STAT1 and 0.78 for phospho-STAT3). Similar Z&#39;-factor values were obtained for the other phospho-STAT assays (STAT4: 0.79; STAT5: 0.78; STAT6: 0.63). A cell-based assay with a Z&#39;-factor &#8805; 0.40 is considered suitable for HTS15. Accordingly, these results demonstrate the robustness of these phospho-STAT assays for HTS applications.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62915/62915fig3v4.jpg" /> 
Figure 3: Detection of phospho-STAT1 (Y701) modulation in U266B1 cells. Cells (200,000 cells/well) seeded in a 96-well culture plate were treated with increasing concentrations of either (A) IFN&#945;2b for 15 min at RT or (B) JAK Inhibitor 1 for 30 min at RT, then with 1 nM (EC80) of IFN&#945;2b for 15 min at RT. Following lysis, lysates were transferred to a low-volume 384-well white plate, followed by the addition of the Antibody Detection Mix. The plate was incubated for 4 h at RT and then read on a TR-FRET-compatible reader. Data are shown as the mean of triplicate wells per assay point. Error bars indicate standard deviation. Some error bars are smaller than the symbol size. Abbreviations: STAT = signal transducer and activator of transcription; IFN = interferon; RT = room temperature; JAK = Janus kinase; TR-FRET = time-resolved F&#246;rster resonance energy transfer; S/B = signal/background ratio. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62915/62915fig3v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62915/62915fig4v4.jpg" /> 
Figure 4: Detection of phospho-STAT3 (Y705) and total STAT3 modulation in HeLa cells. Cells (40,000 cells/well) cultured overnight in a 96-well culture plate were treated with increasing concentrations of either (A) IFN&#945;2b for 20 min at RT or (B) JAK Inhibitor 1 for 30 min at RT then with 1.5 nM of IFN&#945;2b for 20 min at RT. Following media removal and lysis, lysates were transferred to a low-volume 384-well white plate, followed by the addition of the Antibody Detection Mix. The plate was incubated for 4 h at RT and then read on a TR-FRET-compatible reader. Data are shown as the mean of triplicate wells per assay point. Error bars indicate standard deviation. Some error bars are smaller than the symbol size. Abbreviations: STAT = signal transducer and activator of transcription; IFN = interferon; RT = room temperature; JAK = Janus kinase; TR-FRET = time-resolved F&#246;rster resonance energy transfer; S/B = signal/background ratio. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62915/62915fig4v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62915/62915fig5v4.jpg" /> 
Figure 5: Detection of phospho-STAT4 (Y693) modulation in U266B1 cells. Cells (400,000 cells/well) seeded in a 96-well culture plate were treated with increasing concentrations of either (A) IFN&#945;2b for 25 min at 37&#160;&#176;C or (B) JAK Inhibitor 1 for 30 min at RT, then with 1 nM of IFN&#945;2b for 25 min at 37&#160;&#176;C. Following media removal and lysis, lysates were transferred to a low-volume 384-well white plate, followed by the addition of the Antibody Detection Mix. The plate was incubated overnight at RT and then read on a TR-FRET-compatible reader. Data are shown as the mean of triplicate wells per assay point. Error bars indicate standard deviation. Some error bars are smaller than the symbol size. Abbreviations: STAT = signal transducer and activator of transcription; IFN = interferon; RT = room temperature; JAK = Janus kinase; TR-FRET = time-resolved F&#246;rster resonance energy transfer; S/B = signal/background ratio. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62915/62915fig5v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62915/62915fig6v4.jpg" /> 
Figure 6: Detection of phospho-STAT5 (Y694/Y699) and total STAT5 modulation in A431 cells. Cells (75,000 cells/well) cultured overnight in a 96-well culture plate were treated with increasing concentrations of either (A) EGF for 10 min at RT or (B) Erlotinib for 15 min at RT, then with 73 nM of EGF for 10 min at RT. Following media removal and lysis, lysates were transferred to a low-volume 384-well white plate, followed by the addition of the Antibody Detection Mix. The plate was incubated overnight at RT and then read on a TR-FRET-compatible reader. Data are shown as the mean of triplicate wells per assay point. Error bars indicate standard deviation. Some error bars are smaller than the symbol size. Abbreviations: STAT = signal transducer and activator of transcription; EGF = epidermal growth factor; RT = room temperature; TR-FRET = time-resolved F&#246;rster resonance energy transfer; S/B = signal/background ratio. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62915/62915fig6v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62915/62915fig7v4.jpg" /> 
Figure 7: Detection of phospho-STAT6 (Y641) and total STAT6 modulation in HeLa cells. Cells (40,000 cells/well) cultured overnight in a 96-well culture plate were treated with increasing concentrations of either (A) IL-4 for 20 min at RT or (B) JAK Inhibitor 1 for 30 min at RT, then with 0.5 nM of IL4 for 20 min at RT. Following media removal and lysis, lysates were transferred to a low-volume 384-well white plate, followed by the addition of the Antibody Detection Mix. The plate was incubated for 4 h at RT and then read on a TR-FRET-compatible reader. Data are shown as the mean of triplicate wells per assay point. Error bars indicate standard deviation. Some error bars are smaller than the symbol size. Abbreviations: STAT = signal transducer and activator of transcription; IL = interleukin; JAK = Janus kinase; RT = room temperature; TR-FRET = time-resolved F&#246;rster resonance energy transfer; S/B = signal/background ratio. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62915/62915fig7v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62915/62915fig08.jpg" /> 
Figure 8: Detection of phospho-STAT4 (Y693) stimulation in U266B1 cells with the one-plate protocol. Cells (160,000 cells/well) seeded in a low-volume 384-well white plate were immediately treated with increasing concentrations of IFN&#945;2b for 25 min at 37&#160;&#176;C. Following lysis, the Antibody Detection Mix was added directly to the lysate. The plate was incubated overnight at RT and then read on a TR-FRET-compatible reader using a flash lamp or laser excitation. Data are shown as the mean of triplicate wells per assay point. Error bars indicate standard deviation. Some error bars are smaller than the symbol size. Abbreviations: STAT = signal transducer and activator of transcription; IFN = interferon; RT = room temperature; TR-FRET = time-resolved F&#246;rster resonance energy transfer; S/B = signal/background ratio. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62915/62915fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62915/62915fig09.jpg" /> 
Figure 9: Detection of phospho-STAT6 (Y641) stimulation in HeLa cells with the one-plate protocol. Cells (80,000 cells/well) seeded in a low-volume 384-well white plate were immediately treated with increasing concentrations of IL-4 for 20 min at RT. Following lysis, the Antibody Detection Mix was added directly to the lysate. The plate was incubated 4 h at RT and then read on a TR-FRET-compatible reader. Data are shown as the mean of triplicate wells per assay point. Error bars indicate standard deviation. Some error bars are smaller than the symbol size. Abbreviations: STAT = signal transducer and activator of transcription; IL = interleukin; RT = room temperature; TR-FRET = time-resolved F&#246;rster resonance energy transfer; S/B = signal/background ratio. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62915/62915fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/62915/62915fig10v4.jpg" /> 
Figure 10: Intraplate variability study of the phospho-STAT1 (Y701) and phospho-STAT3 (Y705) assays. (A) Phospho-STAT1 assay: suspension cells (200,000 U266B1 cells/well) were treated either with 10 nM of IFN&#945;2b for 15 min or serum-free medium alone (low controls). (B) Phospho-STAT3 assay: adherent cells (20,000 HeLa cells/well) were treated either with 5 nM of IFN&#945;2b for 20 min or with serum-free medium alone. For both assays, the TR-FRET signal was read after 4 h of incubation. Abbreviations: STAT = signal transducer and activator of transcription; IFN = interferon; TR-FRET = time-resolved F&#246;rster resonance energy transfer; S/B = signal/background ratio; CV = coefficient of variation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62915/62915fig10v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Time-resolved FÃƒÂ¶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 ...

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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

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within the chloroplasts, the thylakoid membrane system houses all the photosynthetic pigments, reaction center complexes, and most of the electron carriers, and is responsible for light-dependent ATP synthesis. Over 90% of chloroplast proteins are encoded in the nucleus, translated in the cytosol, and subsequently imported into the chloroplast. Further protein transport into or across the thylakoid membrane utilizes one of four translocation pathways. Here, we describe a high-yield method for isolation of transport-competent thylakoids from peas (Pisum sativum), along with transport assays through the three energy-dependent cpTat, cpSec1, and cpSRP-mediated pathways. These methods enable experiments relating to thylakoid protein localization, transport energetics, and the mechanisms of protein translocation across biological membranes.

We present protocols herein for high-yield isolation of physiologically active thylakoids and protein transport assays for the chloroplast twin arginine translocation (cpTat), secretory (cpSec1), and signal recognition particle (cpSRP) pathways.

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 &#181;L sample at concentrations of &#8805; 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.

Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments.

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

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

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Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis