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Assaying Caspase Activity In Vitro Gavin P. McStay and Douglas R. Green Cold Spring Harb Protoc; doi: 10.1101/pdb.prot080283 Email Alerting Service Subject Categories
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Protocol
Assaying Caspase Activity In Vitro Gavin P. McStay1,3 and Douglas R. Green2 1
Department of Life Sciences, New York Institute of Technology, Old Westbury, New York 11568; 2Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105
Monitoring the activity of a caspase, either as an isolated protein or in a complex mixture (e.g., a cytosolic extract), can be achieved by measuring substrate cleavage. Chromogenic or fluorogenic substrates are available for many caspases. These substrates usually consist of the four-amino-acid motif that is optimal for each caspase and a moiety that, when cleaved, generates either a chromophore or a fluorophore that can be detected using spectrophotometric or fluorimetric means. In this protocol, we describe how to use these substrates to monitor caspase activity in samples containing active caspases (e.g., apoptotic cells). Caspase inhibitors, which contain a moiety that covalently attaches to the active site of the caspase, can be used in these assays. These assays will ascertain whether caspases are involved in a specific process (e.g., whether caspases are activated after an apoptotic stimulus) and are particularly informative if a purified caspase is used. However, the substrates and inhibitors are not specific for a particular caspase in an environment containing multiple caspases. So, if cytosolic or apoptotic cell extracts are used in these assays, additional experiments must be performed to identify exactly which caspases are involved.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPE: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
7-Amino-4-trifluoromethyl coumarin (AFC)-conjugated substrate (EMD Millipore; 20 mM in dimethylsulfoxide [DMSO]) Caspase assay buffer Caspase inhibitor in DMSO (e.g., zVAD-FMK [EMD Millipore] or Q-VD-OPH [MP Biomedicals]) (optional; see Step 2) Source of active caspase (activated extract, apoptotic extract, or purified caspase) To prepare an activated extract from any cell type, see Protocol: Preparation of Cytosolic Extracts and Activation of Caspases by Cytochrome c [McStay and Green 2014]). The extract should be incubated with cytochrome c and dATP for 30 min at 37˚C as described in Steps 18–20 of that protocol before proceeding with Step 1 of the protocol below.
Equipment
96-Well plate (flat bottom, black) 3
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080283
774
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Assaying Caspase Activity In Vitro
Fluorescent plate reader (see Step 4) Software for rate analysis (see Steps 5 and 6) Software may be included with fluorescent plate reader. Microsoft Excel can also be used.
METHOD A flowchart of this protocol is shown in Figure 1.
1. Add 10 µL of caspase-containing sample to each well of a 96-well plate. Ten microliters of sample should equal 100 ng of active caspase, 100 µg of cytochrome c-activated cytosolic extract, or 100 µg of apoptotic lysate. To analyze purified caspase, it may be necessary to use a range of concentrations to account for different levels of caspase activity or variations among preparations. A very active caspase, like caspase-3, may require < 100 ng, whereas a caspase with poor activity, like caspase-9, may require more than this amount.
2. (Optional) Add caspase inhibitor(s) to the sample, and incubate for 10 min at room temperature. Use a range of inhibitor concentrations from 10 to 100 µM. 3. Add caspase assay buffer containing 100 µM of the AFC-conjugated substrate to each sample well. 4. Place the plate in a fluorescent plate reader and incubate at 37˚C. Set the plate reader excitation at 400 nm and emission at 505 nm. Set the plate reader to mix and take a reading every minute for 30 min. 5. Identify the linear portion of the fluorophore-generating reaction and determine its slope, which is the initial rate of the reaction (Fig. 2). The initial rate of the reaction is a measure of the activity of the caspase. There may be an initial lag phase— which represents an equilibration phase in the assay—that should not be included in the analysis of initial velocity. See Troubleshooting.
6. Calculate the specific activity of the caspase by dividing the initial rate of the reaction (from Step 5) by the amount of enzyme. This calculation can be used for a purified caspase. However, the amount of a specific caspase present in a cytosolic extract cannot be accurately determined, and several caspases in an extract may contribute to the activity observed. Thus, for cytosolic extracts, this equation can be used to express activity against a particular substrate. For example, using the approximate values from Figure 2, the activity is 10,000 RFLU/100 μg protein = 0.167 RFLU/sec/μg.
Pipette 10 μL of sample into one well of a 96well plate Preincubate sample with caspase inhibitor if desired for 10 min
Add 100 μL of caspase assay buffer
Place plate in fluorimeter 30 min, 37°C, read every minute with shaking before to each read
Analyze initial velocity of reaction as the measure of caspase activity
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080283
FIGURE 1. Assaying caspase activity in vitro. This procedure takes 1 h to complete.
775
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
G.P. McStay and D.R. Green
25,000 Relative fluorescence light units
Relative fluorescence light units
25,000
20,000
15,000 10,000
5000
20,000
15,000 10,000
0
5000
0 0
20
40
60 Time (min)
80
100
120
0
20
40
60 Time (min)
80
100
120
FIGURE 2. Jurkat cell cytosolic extract (100 µg) was incubated with 100 µM cytochrome c and 1 mM dATP for 30 min at 37˚C. Caspase activity of the activated extract was monitored by following release of AFC from DEVD-AFC. Measurements were taken every 2.5 min with shaking. The graph shows the relative fluorescence light units (RFLU) as a function of time. The right-hand graph indicates the linear portion of the reaction that should be used to determine the rate of the reaction (in red). The gradient of this line gives the value of the rate in change in RFLU/min.
TROUBLESHOOTING Problem (Step 5): The initial rate of reaction is too high to determine. Solution: Dilute samples by factors of 10 and measure the initial rates of these reactions. When two
dilutions are found that are 10-fold different in initial rate, analyze the other samples with these same dilutions. Problem (Step 5): No caspase activity is seen. Solutions: Consider the following.
•
•
Ensure that the scale measuring substrate cleavage is at the appropriate setting. Some programs are set to automatically change the scale during the run, while other programs need to be adjusted manually. To do so, take the equivalent concentration of free fluorophore/chromophore and measure this on the plate reader. Adjust the gain for the reading so that this signal is visible on the chart, but is not above the limits of the newly set gain. Exogenous addition of small molecules or purified proteins to the cytosolic extracts can interfere with apoptosome assembly and downstream caspase activation. This problem generally arises from the addition of additional salt into activation reactions which can interfere with apoptosome assembly (Cain et al. 2001). To avoid interference from these exogenously added molecules dilute them with or dialyze them against homogenization buffer for caspase assays (see Protocol: Preparation of Cytosolic Extract and Activation of Caspases by Cytochrome c [McStay and Green 2014]).
Problem (Step 5): There is no inhibition of caspase activity with caspase inhibitor. Solution: Titrate the active caspase and the inhibitor so that the amount of caspase is within the range
of inhibitor used. DISCUSSION
Analyzing substrate-cleaving activity by fluorimetry yields the caspase activity of a purified caspase, activated extract, or apoptotic extract. Except when using purified caspase, however, these assays are not entirely specific for individual caspases. In a more complex mixture, like a cytosolic extract or an apoptotic cell extract, many caspases are present at the same time. For example, cleavage of an LEHDbased substrate (used as a caspase-9-specific substrate) in an apoptotic cell extract does not necessarily mean caspase-9 has been activated. This substrate is more efficiently cleaved by caspase-3 and caspase776
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080283
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Assaying Caspase Activity In Vitro
6 (McStay et al. 2008). To determine which caspase is responsible for the activity, further examination of the caspases present in the extracts needs to be performed, such as by immunoprecipitation followed by measurements of the activity of the isolated caspases. As with other motif-based tools of caspases, interpretations from results using caspase-specific inhibitors must be made while considering the specific context of the assay and the assay performed. Caspases in isolation will show varying degrees of inhibition by both pan-caspase and caspase-cleavage motif-based inhibitors. However, once these inhibitors are used in assays using cellular extracts or whole cells, problems with interpretation will arise. In situations where multiple caspases are present, the actual caspase inhibited by the inhibitor cannot be inferred directly from the assay. This is the reason for the development of more specific caspase inhibitors (Berger et al. 2006; Edgington et al. 2009, 2012) and also the use of techniques to isolate caspases to determine activity (Tu et al. 2006; McStay et al. 2008). RELATED TECHNIQUES
Caspase inhibitors can be used in cell culture, and even in whole animals. In these cases, the inhibitor must be modified to increase cell permeability, usually by methylating hydrophilic residues, such as aspartic acid. The inhibitor must be incubated for a longer time to ensure uptake by the cell and hydrolysis of the methyl group to produce the active inhibitor. Once the cells have been preincubated with the inhibitor, apoptotic stimuli can be applied. After a certain amount of treatment time, cells or tissues are harvested and analyzed for apoptotic features, such as annexin V staining and caspase immunoblotting, among many other parameters that analyze apoptotic events at the cellular or biochemical level. It is important to ensure that the concentration of caspase inhibitor employed in each assay is sufficient to inhibit executioner caspase-dependent events. RECIPE Caspase Assay Buffer
20 mM PIPES (pH 7.4) 100 mM NaCl 1 mM EDTA 0.1% CHAPS 10% sucrose 10 mM dithiothreitol (add fresh) Store at 4˚C.
ACKNOWLEDGMENTS
Supported by grants from the National Institutes of Health. REFERENCES Berger AB, Witte MD, Denault JB, Sadaghiani AM, Sexton KM, Salvesen GS, Bogyo M. 2006. Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes. Mol Cell 23: 509–521. Cain K, Langlais C, Sun XM, Brown DG, Cohen GM. 2001. Physiological concentrations of K+ inhibit cytochrome c-dependent formation of the apoptosome. J Biol Chem 276: 41985–41990. Edgington LE, Berger AB, Blum G, Albrow VE, Paulick MG, Lineberry N, Bogyo M. 2009. Noninvasive optical imaging of apoptosis by caspasetargeted activity-based probes. Nat Med 15: 967–973. Edgington LE, van Raam BJ, Verdoes M, Wierschem C, Salvesen GS, Bogyo M. 2012. An optimized activity-based probe for the study of caspase-6 activation. Chem Biol 19: 340–352.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080283
McStay GP, Green DR. 2014. Preparation of cytosolic extract and activation of caspases by cytochrome c. Cold Spring Harb Protoc doi: 10.1101/pdb. prot080275. McStay GP, Salvesen GS, Green DR. 2008. Overlapping cleavage motif selectivity of caspases: Implications for analysis of apoptotic pathways. Cell Death Differ 15: 322–331. Tu S, McStay GP, Boucher LM, Mak T, Beere HM, Green DR. 2006. In situ trapping of activated initiator caspases reveals a role for caspase-2 in heat shock-induced apoptosis. Nat Cell Biol 8: 72–77.
777
Assaying Caspase Activity In Vitro Gavin P. McStay and Douglas R. Green Cold Spring Harb Protoc; doi: 10.1101/pdb.prot080283 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Apoptosis Assays (34 articles) Cell Biology, general (1057 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Protocol
Assaying Caspase Activity In Vitro Gavin P. McStay1,3 and Douglas R. Green2 1
Department of Life Sciences, New York Institute of Technology, Old Westbury, New York 11568; 2Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105
Monitoring the activity of a caspase, either as an isolated protein or in a complex mixture (e.g., a cytosolic extract), can be achieved by measuring substrate cleavage. Chromogenic or fluorogenic substrates are available for many caspases. These substrates usually consist of the four-amino-acid motif that is optimal for each caspase and a moiety that, when cleaved, generates either a chromophore or a fluorophore that can be detected using spectrophotometric or fluorimetric means. In this protocol, we describe how to use these substrates to monitor caspase activity in samples containing active caspases (e.g., apoptotic cells). Caspase inhibitors, which contain a moiety that covalently attaches to the active site of the caspase, can be used in these assays. These assays will ascertain whether caspases are involved in a specific process (e.g., whether caspases are activated after an apoptotic stimulus) and are particularly informative if a purified caspase is used. However, the substrates and inhibitors are not specific for a particular caspase in an environment containing multiple caspases. So, if cytosolic or apoptotic cell extracts are used in these assays, additional experiments must be performed to identify exactly which caspases are involved.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPE: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
7-Amino-4-trifluoromethyl coumarin (AFC)-conjugated substrate (EMD Millipore; 20 mM in dimethylsulfoxide [DMSO]) Caspase assay buffer Caspase inhibitor in DMSO (e.g., zVAD-FMK [EMD Millipore] or Q-VD-OPH [MP Biomedicals]) (optional; see Step 2) Source of active caspase (activated extract, apoptotic extract, or purified caspase) To prepare an activated extract from any cell type, see Protocol: Preparation of Cytosolic Extracts and Activation of Caspases by Cytochrome c [McStay and Green 2014]). The extract should be incubated with cytochrome c and dATP for 30 min at 37˚C as described in Steps 18–20 of that protocol before proceeding with Step 1 of the protocol below.
Equipment
96-Well plate (flat bottom, black) 3
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080283
774
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Assaying Caspase Activity In Vitro
Fluorescent plate reader (see Step 4) Software for rate analysis (see Steps 5 and 6) Software may be included with fluorescent plate reader. Microsoft Excel can also be used.
METHOD A flowchart of this protocol is shown in Figure 1.
1. Add 10 µL of caspase-containing sample to each well of a 96-well plate. Ten microliters of sample should equal 100 ng of active caspase, 100 µg of cytochrome c-activated cytosolic extract, or 100 µg of apoptotic lysate. To analyze purified caspase, it may be necessary to use a range of concentrations to account for different levels of caspase activity or variations among preparations. A very active caspase, like caspase-3, may require < 100 ng, whereas a caspase with poor activity, like caspase-9, may require more than this amount.
2. (Optional) Add caspase inhibitor(s) to the sample, and incubate for 10 min at room temperature. Use a range of inhibitor concentrations from 10 to 100 µM. 3. Add caspase assay buffer containing 100 µM of the AFC-conjugated substrate to each sample well. 4. Place the plate in a fluorescent plate reader and incubate at 37˚C. Set the plate reader excitation at 400 nm and emission at 505 nm. Set the plate reader to mix and take a reading every minute for 30 min. 5. Identify the linear portion of the fluorophore-generating reaction and determine its slope, which is the initial rate of the reaction (Fig. 2). The initial rate of the reaction is a measure of the activity of the caspase. There may be an initial lag phase— which represents an equilibration phase in the assay—that should not be included in the analysis of initial velocity. See Troubleshooting.
6. Calculate the specific activity of the caspase by dividing the initial rate of the reaction (from Step 5) by the amount of enzyme. This calculation can be used for a purified caspase. However, the amount of a specific caspase present in a cytosolic extract cannot be accurately determined, and several caspases in an extract may contribute to the activity observed. Thus, for cytosolic extracts, this equation can be used to express activity against a particular substrate. For example, using the approximate values from Figure 2, the activity is 10,000 RFLU/100 μg protein = 0.167 RFLU/sec/μg.
Pipette 10 μL of sample into one well of a 96well plate Preincubate sample with caspase inhibitor if desired for 10 min
Add 100 μL of caspase assay buffer
Place plate in fluorimeter 30 min, 37°C, read every minute with shaking before to each read
Analyze initial velocity of reaction as the measure of caspase activity
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080283
FIGURE 1. Assaying caspase activity in vitro. This procedure takes 1 h to complete.
775
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
G.P. McStay and D.R. Green
25,000 Relative fluorescence light units
Relative fluorescence light units
25,000
20,000
15,000 10,000
5000
20,000
15,000 10,000
0
5000
0 0
20
40
60 Time (min)
80
100
120
0
20
40
60 Time (min)
80
100
120
FIGURE 2. Jurkat cell cytosolic extract (100 µg) was incubated with 100 µM cytochrome c and 1 mM dATP for 30 min at 37˚C. Caspase activity of the activated extract was monitored by following release of AFC from DEVD-AFC. Measurements were taken every 2.5 min with shaking. The graph shows the relative fluorescence light units (RFLU) as a function of time. The right-hand graph indicates the linear portion of the reaction that should be used to determine the rate of the reaction (in red). The gradient of this line gives the value of the rate in change in RFLU/min.
TROUBLESHOOTING Problem (Step 5): The initial rate of reaction is too high to determine. Solution: Dilute samples by factors of 10 and measure the initial rates of these reactions. When two
dilutions are found that are 10-fold different in initial rate, analyze the other samples with these same dilutions. Problem (Step 5): No caspase activity is seen. Solutions: Consider the following.
•
•
Ensure that the scale measuring substrate cleavage is at the appropriate setting. Some programs are set to automatically change the scale during the run, while other programs need to be adjusted manually. To do so, take the equivalent concentration of free fluorophore/chromophore and measure this on the plate reader. Adjust the gain for the reading so that this signal is visible on the chart, but is not above the limits of the newly set gain. Exogenous addition of small molecules or purified proteins to the cytosolic extracts can interfere with apoptosome assembly and downstream caspase activation. This problem generally arises from the addition of additional salt into activation reactions which can interfere with apoptosome assembly (Cain et al. 2001). To avoid interference from these exogenously added molecules dilute them with or dialyze them against homogenization buffer for caspase assays (see Protocol: Preparation of Cytosolic Extract and Activation of Caspases by Cytochrome c [McStay and Green 2014]).
Problem (Step 5): There is no inhibition of caspase activity with caspase inhibitor. Solution: Titrate the active caspase and the inhibitor so that the amount of caspase is within the range
of inhibitor used. DISCUSSION
Analyzing substrate-cleaving activity by fluorimetry yields the caspase activity of a purified caspase, activated extract, or apoptotic extract. Except when using purified caspase, however, these assays are not entirely specific for individual caspases. In a more complex mixture, like a cytosolic extract or an apoptotic cell extract, many caspases are present at the same time. For example, cleavage of an LEHDbased substrate (used as a caspase-9-specific substrate) in an apoptotic cell extract does not necessarily mean caspase-9 has been activated. This substrate is more efficiently cleaved by caspase-3 and caspase776
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080283
Downloaded from http://cshprotocols.cshlp.org/ at TULANE UNIV on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Assaying Caspase Activity In Vitro
6 (McStay et al. 2008). To determine which caspase is responsible for the activity, further examination of the caspases present in the extracts needs to be performed, such as by immunoprecipitation followed by measurements of the activity of the isolated caspases. As with other motif-based tools of caspases, interpretations from results using caspase-specific inhibitors must be made while considering the specific context of the assay and the assay performed. Caspases in isolation will show varying degrees of inhibition by both pan-caspase and caspase-cleavage motif-based inhibitors. However, once these inhibitors are used in assays using cellular extracts or whole cells, problems with interpretation will arise. In situations where multiple caspases are present, the actual caspase inhibited by the inhibitor cannot be inferred directly from the assay. This is the reason for the development of more specific caspase inhibitors (Berger et al. 2006; Edgington et al. 2009, 2012) and also the use of techniques to isolate caspases to determine activity (Tu et al. 2006; McStay et al. 2008). RELATED TECHNIQUES
Caspase inhibitors can be used in cell culture, and even in whole animals. In these cases, the inhibitor must be modified to increase cell permeability, usually by methylating hydrophilic residues, such as aspartic acid. The inhibitor must be incubated for a longer time to ensure uptake by the cell and hydrolysis of the methyl group to produce the active inhibitor. Once the cells have been preincubated with the inhibitor, apoptotic stimuli can be applied. After a certain amount of treatment time, cells or tissues are harvested and analyzed for apoptotic features, such as annexin V staining and caspase immunoblotting, among many other parameters that analyze apoptotic events at the cellular or biochemical level. It is important to ensure that the concentration of caspase inhibitor employed in each assay is sufficient to inhibit executioner caspase-dependent events. RECIPE Caspase Assay Buffer
20 mM PIPES (pH 7.4) 100 mM NaCl 1 mM EDTA 0.1% CHAPS 10% sucrose 10 mM dithiothreitol (add fresh) Store at 4˚C.
ACKNOWLEDGMENTS
Supported by grants from the National Institutes of Health. REFERENCES Berger AB, Witte MD, Denault JB, Sadaghiani AM, Sexton KM, Salvesen GS, Bogyo M. 2006. Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes. Mol Cell 23: 509–521. Cain K, Langlais C, Sun XM, Brown DG, Cohen GM. 2001. Physiological concentrations of K+ inhibit cytochrome c-dependent formation of the apoptosome. J Biol Chem 276: 41985–41990. Edgington LE, Berger AB, Blum G, Albrow VE, Paulick MG, Lineberry N, Bogyo M. 2009. Noninvasive optical imaging of apoptosis by caspasetargeted activity-based probes. Nat Med 15: 967–973. Edgington LE, van Raam BJ, Verdoes M, Wierschem C, Salvesen GS, Bogyo M. 2012. An optimized activity-based probe for the study of caspase-6 activation. Chem Biol 19: 340–352.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080283
McStay GP, Green DR. 2014. Preparation of cytosolic extract and activation of caspases by cytochrome c. Cold Spring Harb Protoc doi: 10.1101/pdb. prot080275. McStay GP, Salvesen GS, Green DR. 2008. Overlapping cleavage motif selectivity of caspases: Implications for analysis of apoptotic pathways. Cell Death Differ 15: 322–331. Tu S, McStay GP, Boucher LM, Mak T, Beere HM, Green DR. 2006. In situ trapping of activated initiator caspases reveals a role for caspase-2 in heat shock-induced apoptosis. Nat Cell Biol 8: 72–77.
777
