Quantification of Protein Kinase Enzymatic Activity in Unfractionated Cell Lysates Using CSox‐Based Sensors

Defining perturbations in protein kinase activity within biological samples can provide insight into disease mechanisms as well as potential targets for drug development. In this article, we present a method that utilizes a phosphorylation‐sensitive amino acid, termed CSox, to afford kinase‐selective biosensors capable of reporting on enzymatic activity directly in biological samples. These sensors produce an increase in fluorescence in response to phosphorylation of an amino acid residue adjacent to CSox. Probes can be designed for either serine/threonine or tyrosine kinases, and analysis can be performed using standard fluorescence equipment. The procedures provided herein represent our optimized protocols for the design, validation, and application of CSox‐based protein kinase activity sensors. Curr. Protoc. Chem. Biol. 6:135‐156 © 2014 by John Wiley & Sons, Inc.


INTRODUCTION
Protein kinases are ubiquitous signaling enzymes that catalyze the transfer of the γphosphoryl group of ATP to a hydroxy-amino acid (Manning et al., 2002). Phosphorylation of a protein substrate can modulate its activity through a variety of mechanisms, including induction of a conformational change or altering affinity toward protein binding partners. The >500 protein kinases encoded in the human genome regulate a variety of signaling pathways such as cell growth, cell survival, inflammation, metabolism, and cell migration. As a consequence, the dysregulation of kinase enzymatic activity can drive a variety of disease phenotypes. These observations have resulted in a concerted effort to develop kinase inhibitors for clinical applications (Cohen, 2002). Beyond the utility of small-molecule protein kinase inhibitors in the clinic, chemical genetics has emerged as a powerful tool to elucidate the biological function of a kinase of interest (KOI;Bishop et al., 2000). However, complementary methodologies to quantitatively assess the enzymatic activity of a KOI in biological samples containing numerous endogenous kinases have lagged behind. This has led to the standard practice of using activity proxies to infer kinase activity in biological samples.
Previously, we described an approach to directly monitor kinase activity in unfractionated cell lysates utilizing a phosphorylation-sensitive fluorophore termed Sox . In this article, we present our most up-to-date usage of this fluorophore, which involves covalent linkage to an engineered cysteine within a synthetic kinase substrate (Lukovic et al., 2008). These CSox-based sensors are capable of detecting phosphorylation through a process known as chelation-enhanced fluorescence (CHEF, Fig. 1A). Importantly, this approach allows the user to tune the selectivity of the CSox-based substrate for the KOI and directly monitor the activity of that KOI in the presence of the endogenous kinome Stains et al., 2011;Fig. 1B).
The Basic Protocol provides our optimized procedures for utilizing CSox-based kinase activity sensors to quantify kinase signaling changes in cell lysates derived from samples treated with an external stimulus. This procedure relies on careful optimization of the kinase activity probe (Support Protocol 1) as well as validation of sensor selectivity in the presence of endogenous kinases (Support Protocol 2). Detailed procedures for the chemical synthesis of CSox-based activity sensors have been described elsewhere (Pearce et al., 2001;Lukovic et al., 2008). Herein we provide abbreviated procedures for the synthesis of Sox-Br, a key intermediate required for the construction of CSox-based activity sensors, as well as alkylation of synthetic peptide substrates with Sox-Br (Support Protocol 3). Alternatively, CSox-based activity sensors are currently available under the Omnia trademark from Life Technologies and can be custom synthesized to user specifications. Taken together, these resources allow for the successful design, synthesis, validation, and application of CSox-based activity sensors by any lab.

STRATEGIC PLANNING
An overall flowchart of the protocols described in this unit is shown in Figure 2.

Designing and Evaluating Initial Sensor Constructs
Sequences for initial sensor designs can be drawn from the primary literature. However, since the selectivity of CSox-based sensors is largely dependent on the substrate sequence, careful attention should be given to any available information concerning the kinetic parameters as well as selectivity for the KOI. Consensus phosphorylation sites often do not provide adequate selectivity among endogenous kinases; well-characterized substrates should be employed when possible. As a general guideline, we have found that peptide substrates with k cat values of >20 min −1 provide a sufficient turnover in order to visualize endogenous kinase activity in cell lysates within 1 hr (Stains et al., 2012). In addition, K M values in the low μM range (<5 μM) typically provide selectivity for the KOI while minimizing off-target activity if appropriate sensor concentrations are used (i.e., 1× to 2× K M ). Once a substrate sequence is chosen, the position of CSox within the substrate as well as the in vitro assay conditions should be optimized (Support Protocol 1). Substrates should be evaluated against recombinant kinases with known overlapping sequence specificity. If off-target activity is observed at this stage, the culprit kinase can be easily identified, since the reactions are run in parallel with each recombinant enzyme, and appropriate small-molecule inhibitors can be added to the assay. These initial assays allow for the optimization of reaction conditions before transitioning to more complex systems such as cell lysates.

Validation of Sensors in Cell Lysates
Following in vitro assay optimization, the selectivity of CSox-based activity sensors should be evaluated in cell lysates containing endogenous kinases. First, the amount of lysate used in the assay is optimized to provide the highest signal-to-noise between samples stimulated for KOI activity and controls. Following this step, the KOI is immunodepleted from the lysate, allowing for the contribution of the remaining endogenous kinases to be determined. Antibodies that are compatible with immunoprecipitation should be utilized for this experiment. Lastly, known small-molecule inhibitors of the KOI can be employed to further delineate sensor selectivity. If possible, multiple inhibitors should be employed to establish that signal from the sensor is not the result of an off-target kinase that is also inhibited by the chosen small molecule.

GENERAL CELL LYSATE ASSAY FORMAT
The hallmark of a validated CSox-based kinase activity sensor is its ability to selectively report on perturbations in kinase activity in complex biological samples, such as unfractionated cell lysates. Kinase activity can be stimulated according to standard protocols for the KOI. Once lysate samples are obtained, alterations in the enzymatic activity of a KOI can be determined using a fluorescence plate reader by analysis of the increase in fluorescence of the CSox-based kinase activity sensor with respect to time.

Materials
Appropriate adherent mammalian cell line (e.g., ATCC) Cell culture medium appropriate for cell line used Stimulation method of choice for the KOI Phosphate-buffered saline (PBS; Life Technologies, cat. no.10010-023), ice cold Nondenaturing lysis buffer (see recipe) Liquid nitrogen in Dewar flask 10× Mg 2+ assay buffer (see recipe, the concentration of Mg 2+ is optimized in Support Protocol 1) 10× CSox peptide sensor solution (see recipe, the optimal concentration of CSox-based sensor is determined in Support Protocol 1) 10× ATP solution (10 mM, see recipe) Corning 150-mm culture dishes (Sigma, cat. no. CLS430599) Cell scrapers (Fisher Scientific,  3. Maintain the second dish under normal growth conditions to serve as a nonstimulated control.
4. After stimulation, place both dishes on ice, remove medium and rinse three times, each time with 10 ml ice-cold PBS, making sure to remove all liquid following each rinse.
5. Add 400 μl of ice-cold nondenaturing lysis buffer to the dish and, using a cell scraper, vigorously spread the lysis buffer across the entire surface.
6. Angle the dish slightly downward and begin scraping the lysate toward the bottom of the dish, making sure to scrape the entire surface of the dish.
7. Collect the accumulated lysate from the bottom of the dish with a micropipettor and place in a 1.6-ml microcentrifuge tube on ice.
8. Mark each sample as "control" or "stimulated" as appropriate.
9. Incubate both sets of lysate on ice for 15 min.
10. Microcentrifuge samples for 5 min at 10,000 × g, 4°C, to pellet the insoluble material. Carefully collect the soluble fraction, transfer to a labeled microcentrifuge tube, and place on ice.
Certain cell lines produce a noticeable lipid layer that will appear as a cloudy material on top of the soluble fraction. If a significant lipid layer is present, pierce the material with a pipet tip to recover the soluble fraction.
11. Aliquot each sample into 20-μl fractions in microcentrifuge tubes on ice.
12. Flash freeze the aliquotted samples in liquid nitrogen and store at −80°C.
For kinases studied thus far, we have not detected any appreciable loss of activity over a 6-month period in this lysis buffer formulation. However, if long-term storage is desired, the stability of kinase enzymatic activity in lysis buffer should be determined.
Acquire and analyze data 13. Turn on the fluorescence plate reader and allow the instrument to auto-calibrate.
The initial 10-min read without the addition of lysate allows the user to define the starting fluorescence of the CSox probe. This data can be used to identify reactions that have reached completion after addition of lysate but prior to reading on the plate reader (generally observed as a flat line with a total fluorescence >2-fold higher than the initial read).

Read time can be increased or reduced based on individual kinase kinetics.
15. Make a master mix consisting of water, 1× Mg 2+ assay buffer, 1× CSox peptide sensor (exact concentrations will vary; see below), and 1× ATP. 16. Aliquot an appropriate amount of master mix into individual wells of the 96-well half-area plate, taking into account the cell lysate solution to be added later in order to reach the final volume of 120 μl.
For example, in an assay of 120 μl total volume of which 6 μl would be cell lysate, one would add 114 μl of master mix to each well.
17. Place the 96-well plate into the fluorescence plate reader and begin the kinetic protocol.
18. Remove the appropriate number of lysate samples from the −80°C freezer and place on ice.
Avoid subjecting samples to freeze/thaw cycles, as this can decrease kinase activity and lead to inconsistent data. Freeze/thawed samples may be used for western blotting or other experiments that are not reliant on preserved kinase activity.
19. Dilute the lysate samples with nondenaturing lysis buffer to the appropriate concentration as previously determined (see Support Protocol 2). Keep samples on ice.
20. Following the initial 10 min of thermal equilibration and 10-min kinetic read without lysate, remove the 96-well plate from the fluorescence plate reader and add the appropriate type and amount of lysate dilutions to the desired wells.
21. Mix the assay solution in each well multiple times with a multichannel pipettor.
The presence of lysis buffer in the assay solution can lead to the formation of bubbles in the assay wells. Bubbles must be removed prior to data collection, as they can interfere with optical assays. Large bubbles may be readily dissipated with a hypodermic needle; however, small bubbles are extremely difficult to remove. Avoid introducing small bubbles to the assay wells by not injecting air into the assay solution while mixing with the multichannel pipettor.
22. Place the 96-well plate back into the fluorescence plate reader and initiate the 2-hr data-collection phase.
23. Following data collection, remove the 96-well plate from the plate reader and cover with Parafilm to protect from dust or other contaminants.

96-well plates may be used in multiple experiments, but individual assay wells should not be reused.
24. Plot the collected data for each assay well as a function of Arbitrary Fluorescence Units (AFU) and time (min) in Microsoft Excel or other preferred spreadsheet program.
25. Select the time points for the initial, linear portion of data and fit to a trend line.
The entire data set should graph as a hyperbolic plot. However, only the initial, linear portion of the plot should be analyzed. There are two situations that can arise in which obtained data may not produce a hyperbolic plot. First, the data may exhibit an initial lag phase prior to the linear region of the data that should be used for work-up Lukovic et al., 2009;Stains et al., 2011Stains et al., , 2012. In this case, the initial lag phase should not be included in the analysis, as this will bias the fit. Second, the data may exhibit a linear, positive slope but may not become asymptotic. This occurs when the assay has not been monitored for a sufficient time to allow for the observation of the asymptotic portion of the data. This does not pose a problem, as the initial linear portion of the plot represents the data of interest and the reaction rate can be obtained without the asymptotic portion. In all data work-up, it is important to determine the reaction slope over the same time period for each assayed sample.
26. Find the average reaction slope (slope of trend lines from step 25) and calculate the standard deviation of each triplicate sample set. The average reaction slope is directly proportional to the enzyme activity.
27. Plot the average reaction slopes of each sample type as a bar graph with error bars consisting of the standard deviation of each sample set.

DESIGN AND EVALUATION OF INITIAL SENSOR CONSTRUCTS
The initial design, characterization, and in vitro evaluation of potential substrates are essential steps in the development of optimized CSox-based kinase activity sensors. Following the design and synthesis of potential substrates, certain physical properties of the sensor (Mg 2+ K D , fold fluorescence signal increase) and kinetic parameters of the enzyme/substrate complex (K M , V max ) should be determined in order to guide further assay optimization. Furthermore, identification of off-target interactions between the sensor and highly homologous kinases in a controlled, in vitro environment provides for troubleshooting opportunities prior to analyzing the relatively more complex reaction conditions encountered in cell lysate assays.

Materials
Potential kinase peptide substrates (synthesize as described in Support Protocol 3 or purchase from Life Technologies) 0.1 M NaOH containing 1 mM disodium EDTA 500 mM (10×) Tris·Cl (pH = 7.5 at 22°C) 1.5 M (10×) NaCl MgCl 2 (Alfa Aesar, cat. no. 10797) 10× Mg 2+ assay buffer (see recipe, the concentration of Mg 2+ is optimized in step 11 of this protocol) 10× ATP solution (10 mM 2. Design four peptides as pairs in which each peptide pair is delineated by a cysteine substitution at the (+)2 or (-)2 amino acid relative to the site of phosphorylation.
Within each set of peptides, one sequence should contain the native amino acid that will serve as the site of phosphorylation (this will be the substrate, or nonphosphopeptide) and the other sequence should contain the corresponding phosphoamino acid at the site of phosphorylation (this phosphopeptide sequence will serve as a positive control 3. Synthesize the four peptides according to Support Protocol 3 (Pearce et al., 2001;Lukovic et al., 2008) or obtain the sequences commercially from Life Technologies.
4. Dissolve the purified peptide in water. Determine the peptide concentration by diluting a small aliquot of the peptide in 0.1 M NaOH containing 1 mM disodium EDTA, and measuring absorbance at 355 nm (ɛ 355 = 8247 M −1 cm −1 ) .

Equation 1
The K D of the phosphopeptide for Mg 2+ should be tighter than the K D of the corresponding nonphosphopeptide for Mg 2+ . This allows for the discrimination of the phosphorylation state of the target amino acid using the appropriate concentration of Mg 2+ , which is determined below.
corresponding phosphopeptide for Mg 2+ within each pair (in a total of 120 μl per well).
This assay should be performed in triplicate for each concentration of Mg 2+ , yielding a total of 9 wells for each peptide.
10. Analyze the assay wells with the fluorescence plate reader using the procedure described in step 5. 14. Add 108 μl of each master mix to individual wells.
15. Make separate master mix solutions of water, Mg 2+ assay buffer, and ATP that will yield final concentrations of 1 μM, 2.5 μM, and 5 μM phosphopeptide (reaction product) respectively for both peptide pairs 1 and 2 as described in step 13.
Assays with phosphopeptides should also be performed in triplicate.
16. Add 108 μl of each master mix type from step 15 to individual wells.

Equation 2
This plot yields values for K M (μM) and V max (μM/min). k cat (min −1 ) can be calculated by dividing V max by the concentration of the KOI in μM.
29. The specificity constant, k cat /K M (μM −1 min −1 ), can be used to compare the catalytic efficiency of each substrate.
In general, substrates with larger values of k cat /K M should be used for further assays.

Refer to the Strategic Planning section for additional details concerning interpretation of kinetic parameters for subsequent assays.
Kinase panel assay 30. Use the literature or sequence homology (Manning et al., 2002) to identify a panel of kinases with potential overlapping substrate specificity with the KOI.
31. Make a master mix with water, Mg 2+ assay buffer, ATP, and the optimal substrate determined above. Use a final substrate concentration that is at least 1× to 2× K M to achieve a relatively high rate of turnover during the assay. Assume that you will add 12 μl of kinase to each well to reach a final volume of 120 μl. Assay each kinase in triplicate.

VALIDATION OF SENSORS IN CELL LYSATES
In order to validate CSox-based kinase activity sensors, it is necessary to optimize the amount of lysate to be used in assays and demonstrate selectivity in cell lysates derived from the cell line of interest. Two methods are presented to determine activity due to off-target phosphorylation, immunodepletion, and assays in the presence of selective inhibitors of the KOI.

Materials
Cell lysates (Basic Protocol 1) BioRad protein assay dye reagent concentrate (BioRad, cat. no. 500-0006) Nondenaturing lysis buffer (see recipe) 10× Mg +2 assay buffer (see recipe, the concentration of Mg 2+ is optimized in Support Protocol 1) CSox peptide substrate (optimal sequence identified from Support Protocol 1) 10× ATP solution (10 mM, see recipe) Anti-KOI antibody (appropriate for immunoprecipitation) Naïve IgG antibody (isotype matched to anti-KOI antibody) Protein A-or G-Sepharose (e.g., GE Life Sciences) 100× stock(s) of small-molecule inhibitor(s) of the KOI Cell culture-grade DMSO (e.g., Sigma) Fluorescence plate reader (SynergyH2 or similar) Corning 96-well half-area flat-bottom white microplates (Sigma, cat. no. CLS3693) Multichannel pipettor Orbital rocker/shaker Nonlinear regression analysis software (e.g., KaleidaGraph) Additional reagents and equipment for kinase activity measurements (Basic Protocol 1) Optimize lysate amount in assay 1. Following the preparation of cell lysates (see Basic Protocol 1), thaw the lysates on ice and quantify protein content using the BioRad protein assay according to the manufacturer's instructions.
2. Dilute each lysate with nondenaturing lysis buffer to a final concentration of 4 μg/μl protein.

Kinase Activity Using CSox-Based Sensors 145
Current Protocols in Chemical Biology Volume 6 3. Prepare serial dilutions of each lysate in lysis buffer to final concentrations of 4, 2, 1, 0.4, and 0 μg/μl (in a total of 85 μl each).
4. Prepare a master mix containing water, Mg 2+ assay buffer, CSox peptide substrate, and ATP as described in step 15 of Basic Protocol 1. Assume that you will add 25 μl of lysate to each well (i.e., 95 μl mix per well) and will evaluate each sample in triplicate.
5. Set up the plate reader protocol following Basic Protocol 1, step 14.
6. Aliquot the master mix from step 4 into a 96-well plate (95 μl in each well). Place in the plate reader and begin the kinetic protocol.
7. Following the temperature equilibration and initial read portion of the protocol, pipet 25 μl of each lysate into its designated well.
8. Mix the contents of each well with a multichannel pipettor.
9. Place the plate back in the plate reader and continue the kinetic protocol.
10. Following the kinetic protocol, plot AFU versus time for each lysate.
11. Fit the linear portion of each plot; this corresponds to the relative enzyme activity for each sample.
12. Compare the amount of time each plot is linear and the average slope for each sample.
The amount of lysate used in subsequent assays should provide a substantial linear range (i.e., >5 min) and should allow for differences in enzyme activities in control versus stimulated samples to be observed.

Perform immunodepletion
13. Using the stimulated lysates from the steps above, incubate 600 μg of lysate with 20 μl of Protein A-or Protein G-Sepharose beads, with gentle shaking for 1 hr at 4°C in a microcentrifuge tube.
The choice of Protein A-or Protein G-Sepharose will depend on the anti-KOI antibody used (Hage, 2006). 14. Centrifuge the sample 5 min at 1000 × g, 4ºC, and transfer the pre-cleared lysate to two new microcentrifuge tubes (divide the sample in half, by volume).
15. Add 1 to 5 μg of anti-KOI antibody to one lysate sample and the same amount of naïve IgG to the other sample.
The naïve antibody will serve as a negative control, since the KOI should not be depleted. 20. Run kinase activity measurements on the input (non-treated lysate), immunodepleted, and naïve IgG control samples using Basic Protocol 1 with the optimal conditions identified above.

Determine the slopes for each of the samples.
The residual activity in the immunodepleted sample will provide information regarding activity due to off-target kinases and should be compared to western blots to determine the extent of depletion of the KOI. Alternatively, if the K M of the KOI for ATP is known the K I of the inhibitor can be calculated using the Cheng-Prusoff equation and compared to the calculated K I obtained from the CSox assay.

If the inhibitor is ATP-competitive and the reported IC 50 value was obtained under similar concentrations of ATP, a large deviation from the reported value could indicate sensor phosphorylation by off-target kinases.
Kinase Activity Using CSox-Based Sensors 147 Current Protocols in Chemical Biology Volume 6 34. In the plot from step 33, take note of the percent residual activity at the highest inhibitor concentration.
If there is >10% residual activity, this could also indicate sensor phosphorylation by offtarget kinases. For approaches to mitigate off-target kinase activity, see Troubleshooting.

SOX-Br SYNTHESIS AND ON-RESIN ALKYLATION
Procedures for the synthesis of 2-bromo-8-tert-butyldiphenylsilyloxy-5-(N,Ndimethyl)sulfonamidoquinoline (Sox-Br, Fig. 3) and the alkylation of cysteine residues within kinase peptide substrates are given below (Pearce et al., 2001;Lukovic et al., 2008). Synthesis of Sox-Br will require knowledge of standard organic chemistry techniques and basic organic synthesis laboratory equipment. This procedure also assumes a general knowledge of Fmoc SPPS (Chan and White, 2000).
3. Transfer reaction mixture to a 2-liter separatory funnel containing a slurry of brine/ice (500 ml) and CH 2 Cl 2 (500 ml). Shake the separatory funnel and allow the layers to separate. Dry the organic layer over 10 g of potassium carbonate.
7. Allow the reaction to stir for an additional 10 min.
8. Remove the solvent via rotary evaporation. Redissolve the residue with 50 ml of CH 2 Cl 2 and remove solvent via rotary evaporation. Repeat two additional times to remove residual dimethylamine.
11. Allow the reaction to stir under N 2 for 2 hr.
12. Dilute the reaction with 250 ml of EtOAc and wash once with 50 ml saturated ammonium chloride, then twice, each time with 50 ml brine, in an appropriately sized separatory funnel. Dry the organic layer over MgSO 4 .
16. Allow the reaction to stir for 17 hr at 95°C.
17. Cool the reaction to 25°C and filter the reaction mixture through Celite. Wash the Celite with additional dioxane (30 ml).
18. Remove the solvent via rotary evaporation and redissolve the residue in EtOAc (900 ml).
23. Add the solution of from step 22 dropwise to the solution from step 21.
24. Allow the reaction to stir for 15 min.
25. Dilute the reaction mixture with diethyl ether (800 ml) and wash with once with saturated ammonium chloride (200 ml), twice with water (100 ml), and once with brine (100 ml). Dry the organic layer over MgSO 4 .
30. Stir the reaction for 4 hr at 0°C. 31. Dilute the reaction mixture with diethyl ether (1200 ml) and wash the organic layer with water (150 ml) and brine (100 ml). Dry the organic layer over MgSO 4 .
Product identity and purity should be determined by MS and NMR, referring to previously published spectra .
The final product is a yellow oil that generally contains an appreciable amount of residual (4). However, (4) does not interfere with the alkylation reaction and can be easily washed away after resin-based coupling of (7) to the peptide.

On-resin alkylation of peptides with Sox-Br
33. Following SPPS (Stawikowski and Fields, 2012) of the designed substrate sequence with Cys(Mmt) at the site of Sox incorporation (Support Protocol 1), weigh 20 μmol of the dried peptide on resin, and transfer to a 3-ml syringe with frit.
The resin should be between the frit and plunger.
34. Swell the resin five times, each time for 5 min in 2 ml CH 2 Cl 2 with agitation.

REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes.

Critical Parameters
When the goal is using CSox-based sensors to probe kinase activity in biological samples, the selectivity of the substrate sequence and the corresponding sensor are of critical importance. If the selectivity of the chosen substrate is not defined, sources of off-target activity can be identified by employing a panel of recombinant enzymes (Support Protocol 1) as well as immunodepletions and inhibitor assays in cell lysates (Support Protocol 2). Sources of off-target activity identified in vitro can be addressed by employing known inhibitors for the identified off-target kinase in order to suppress background signal from off-target kinase ac-tivity. The suppression of unknown off-target activity in cell lysates requires more careful optimization. In this case, two strategies can be pursued. If a selective inhibitor for the target kinase is known, this can be used to background-subtract the effects of off-target kinases (i.e., one can run assays in the presence or absence of inhibitor and subtract offtarget signal remaining in the inhibitor-treated samples). Alternatively, the known promiscuity of kinase inhibitors (Davis et al., 2011) can be exploited to suppress the activity of entire families of potential off-target enzymes.
As a complementary approach, methods have been described for the high-throughput optimization of peptide substrates for a KOI (Gonzalez-Vera et al., 2009). These approaches can be employed to afford catalytically efficient peptide substrates that can be translated into CSox-based probes, as described in the protocols above. In addition, if KOI-specific targeting domains are known,  these can be appended to CSox-based peptides to further increase KOI selectively Stains et al., 2011).

Troubleshooting
A troubleshooting guide for the protocols in this unit is presented in Table 1.

Anticipated Results
The successful application of these protocols should afford a selective CSox-based kinase activity sensor that can be used to assess perturbations in kinase activity in biological samples. Lysates prepared using the conditions outlined in Basic Protocol 1 should produce differing rates of phosphorylation of the CSoxbased sensor that reflect endogenous activity of the KOI (Fig. 4). Using these procedures, we have generated several CSox-based activity probes capable of detecting endogenous kinase activity in cell lysates and tissue homogenates Stains et al., 2011Stains et al., , 2012.

Time Considerations
Sox-Br-, and CSox-containing peptide sequences can be generated in 3 to 4 weeks (Support Protocol 3). Initial validation of the substrate peptide, determination of fluorescent properties, kinetic parameters of phosphoryla-tion, and in vitro kinase panel screens can be performed in 1 to 2 weeks (Support Protocol 1). The individual kinase activity measurement can be performed in a matter of hours. Further validation of the sensors in cell lysates can be more time consuming, given the requirement to culture cells, but can be achieved in 3 to 4 weeks provided that a suitable cell line and stimulation condition for the KOI is known (Support Protocol 2).