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Detection of Caspase Activity Using Antibody-Based Techniques Gavin P. McStay and Douglas R. Green Cold Spring Harb Protoc; doi: 10.1101/pdb.prot080291 Email Alerting Service Subject Categories
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Protocol
Detection of Caspase Activity Using Antibody-Based Techniques 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
A number of antibodies have been generated that recognize caspases from mammalian model organisms. These include antibodies that recognize specific caspase pro-forms and others that bind caspase cleavage fragments. These antibodies are excellent reagents for identifying which executioner caspases have been activated following application or induction of a specific apoptotic stimulus. This approach is more difficult to use with initiator caspases, however, because cleavage does not necessarily correlate with caspase activation. In this protocol, cultured cells are treated with a proapoptotic stimulus, and then protein lysates are prepared from the treated cells. The proteins are then separated by gel electrophoresis and transferred to a suitable membrane. The fragment-specific antibodies that recognize executioner caspases are used in a western analysis to determine the extent of activation and to aid in identifying which caspases have been activated.
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. RECIPES: 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
Antibody to caspase, recognizing either full-length protein or a cleavage fragment Antibody recognizing primary antibody, conjugated to horseradish peroxidase Bradford protein assay materials (see Step 6) Cell line to be treated with an apoptotic stimulus Dry ice–methanol bath (optional; see Step 4) Enhanced chemiluminescent reagent (Thermo Scientific) Homogenization buffer for caspase activation (optional; see Step 4) Nonfat dry milk (5%) in TBS-T NP-40 lysis buffer Add protease inhibitors (e.g., complete protease inhibitor cocktail tablets [Roche]) to the NP-40 lysis buffer according to the manufacturer’s instructions.
Pan-caspase inhibitor (e.g., methylated zVAD-FMK) PBS (A) 3
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080291
783
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
G.P. McStay and D.R. Green
Proapoptotic stimulus The apoptotic stimulus can be a reagent like etoposide, a ligand like anti-Fas antibody, or a physical perturbation like UV radiation.
SDS–PAGE running buffer SDS–PAGE sample buffer (4×) TBS-T buffer (e.g., Abcam or Cell Signaling Technology) Trypsin–EDTA (e.g., trypsin–EDTA in PBS [Life Technologies]) Western transfer buffer Equipment
Autoradiography film, film cassette, and film processor Centrifuge (benchtop) for 15-mL conical tubes Heating block set to 95˚C Microcentrifuge at 4˚C Nitrocellulose membrane SDS–PAGE equipment Western transfer apparatus METHOD A flowchart of this protocol is shown in Figure 1.
1. Treat cells with a proapoptotic stimulus. Inclusion of negative controls is important at this stage. Include two types of negative controls: (1) cells not treated with the proapoptotic stimulus and (2) cells pretreated with a pan-caspase inhibitor that is able to enter cells (e.g., methylated zVAD-FMK). In the latter control, incubate cells and the pan-caspase inhibitor for 1 h before treatment with proapoptotic stimulus. The concentration of the inhibitor depends on which inhibitor is included (e.g., use 100 µM methylated zVAD-FMK or 20 µM QVD-OPH).
Harvest treated cells and wash with PBS Ensure all floating/apoptotic cells are harvested Lyse cells in NP-40 lysis buffer with protease inhibitors
Centrifuge, keep supernatant 15,000g, 10 min, 4°C Determine protein concentration Depends on laboratory preferences Add SDS-PAGE sample buffer to sample 95°C, 10 min Run 20 μg of the heated sample on SDS-PAGE Conditions depend on laboratory preferences Western transfer Conditions depend on laboratory preferences Immunoblot with caspase-specific antibody and secondary antibody Detect protein: antibody complexes using chemiluminescence
784
FIGURE 1. Detection of caspase activity using antibody-based techniques. This protocol requires 1–1.5 d to complete. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080291
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Detecting Caspase Activity with Antibodies
2. Harvest treated cells by collecting the medium (containing detached apoptotic cells) and releasing adherent cells from the dish with trypsin–EDTA solution. Combine all of the cells in a centrifuge tube. Collect the cells by centrifugation at 400g for 5 min at room temperature. 3. Wash the cells in PBS and collect them by centrifugation as in Step 2. 4. Discard the supernatant. Add 100 µL of NP-40 lysis buffer with protease inhibitors per 5 × 105 cells. Lyse the cells by pipetting the cell pellet up and down until no clumps remain. Incubate the cells on ice for 10 min. Alternatively, cytosolic extracts can be prepared by resuspending cell pellets in homogenization buffer for caspase activation, followed by three freeze–thaw cycles in a dry ice-methanol bath.
5. Centrifuge the lysate at 15,000g for 10 min at 4˚C.
6. Determine the protein concentration of the supernatant using the Bradford assay or another suitable method. 7. Add 4× SDS–PAGE sample buffer to an appropriate volume of cell lysate containing 20 µg of protein. Heat at 95˚C for 10 min.
8. Run the heated lysate on a 12% SDS–PAGE gel at 200 V until the gel front dye reaches the bottom of the gel. 9. Transfer the separated proteins onto nitrocellulose using standard western transfer techniques and western transfer buffer. 10. Block the membrane with 5% nonfat dry milk in TBS-T for 30 min at room temperature. Rock the membrane gently during blocking. 11. Wash the membrane in TBS-T. Incubate the membrane in primary antibody (dilution depends on the antibody being used) in 5% nonfat dry milk in TBS-T solution for 2 h, rocking at room temperature (or overnight at 4˚C). 12. Remove the antibody solution. Wash the membrane three times for 5 min each by rocking in TBS-T at room temperature.
13. Incubate the membrane in secondary antibody (1:10,000 dilution) in 5% nonfat dry milk in TBST solution for 2 h with rocking at room temperature. 14. Remove the antibody solution. Wash the membrane three times for 5 min each by rocking in TBS-T at room temperature. 15. Expose the membrane to enhanced chemiluminescent reagent for 1 min. 16. Cover the membrane with plastic and expose to X-ray film for different amounts of time (seconds to minutes) to detect bands corresponding to full length and cleaved caspases. Other methods for chemiluminescence capture are suitable, such as a gel documentation system that has chemiluminescent detection capability. The exposed western blot should show smaller protein bands corresponding to the cleaved form of the caspases (15 kDa–20 kDa for executioner caspases and 40 kDa for initiator caspases). If these are true caspase cleavage products, then the bands should be absent from both the untreated cells and the cells pretreated with pan-caspase inhibitor before treatment with proapoptotic stimulus (Fig. 2). See Troubleshooting.
TROUBLESHOOTING Problem (Step 16): No cleaved caspases are seen in apoptotic extracts. Solution: Some caspases (such as caspase-7 and caspase-9) once cleaved become susceptible to deg-
radation in an ubiquitin-dependent manner. To improve the detection of these cleavage products, take samples at early time points (several hours instead of overnight treatment). It is also possible to use a proteasome inhibitor, such as MG132, to prevent degradation. There are also some Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080291
785
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
G.P. McStay and D.R. Green
37 Pro-caspase-3 25
FIGURE 2. Caspase-3 western blot of Jurkat cells preincubated with 100 µM zVAD-FMK for 10 min before the induction of apoptosis overnight. The untreated samples show caspase-3 as full-length procaspase at 30 kDa. In the apoptotic sample there is less full-length caspase-3 and a 15 kDa band appears, representing cleaved caspase-3 fragments. In cells preincubated with zVAD-FMK before treatment with the proapoptotic stimulus, only full-length caspase-3 is seen, indicating that cleavage was caspase dependent.
Cleaved caspase-3
15 –
+
–
+
zVAD-FMK
Ap
Un
op
tre
to
at
tic
ed
circumstances in which caspases remain uncleaved when apoptosis is triggered. This is especially true for initiator caspases (see Discussion). DISCUSSION
Detection of caspase cleavage products by western blotting is a straightforward method for implicating a specific caspase in a particular death pathway. However, this approach is crude, because it does not take into account the feedback that occurs in apoptotic pathways or the mechanisms of activation of initiator caspases. Monitoring cleavage events of executioner caspases provides a direct read-out of activation. The disappearance of the full-length protein, coupled with the appearance of the cleaved fragments containing the large and small catalytic subunits, indicates that one or more executioner caspases have been activated (Fig. 3). The same methodology does not yield comparable results with initiator caspases. Although cleavage of an initiator caspase is sometimes necessary for its activity, it is not sufficient to activate it. Thus, simply monitoring the cleavage of an initiator caspase does not tell us whether the caspase dimerized—and was therefore truly activated—before cleavage. In general, however, it is true that if an initiator caspase is not cleaved, then it is unactivated (Fig. 3). Cleaved initiator caspases typically function as substrates of executioner caspases as part of the controlled demolition of a cell (Inoue et al. 2009).
RELATED TECHNIQUES
Antibodies that recognize specific cleaved fragments of caspases are available, and these are commonly used in ELISAs or immunohistochemical staining of tissue to detect active caspases.
Initiator
1
2
Executioner
3
4
5
6
Full length Cleaved
Active
786
?
?
?
No
Yes
Yes
FIGURE 3. Schematic representation of a caspase immunoblot depicting problems of interpreting activity based on caspase cleavage patterns. Initiator caspases in lanes 1, 2, and 3, whether uncleaved, cleaved, or a mixture, do not reveal whether the caspase was activated. Executioner caspases in lanes 4, 5, and 6 show that uncleaved executioner caspases are not active and cleaved executioner caspases are active.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080291
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Detecting Caspase Activity with Antibodies
RECIPES Homogenization Buffer for Caspase Activation
10 mM HEPES (pH 7.0) 5 mM MgCl2 0.67 mM DTT 1 protease inhibitor cocktail tablet (Roche) for the appropriate volume of homogenization buffer The homogenization buffer without DTT or protease inhibitors can be stored at –4˚C for up to 6 mo. Once DTT and protease inhibitors are added, the homogenization buffer should be stored at –20˚C in 10-mL aliquots; it will last for up to 1 yr under these conditions. NP-40 Lysis Buffer
NaCl (150 mM) NP-40 (1.0%) Tris-Cl (50 mM, pH 8.0) For 1 L of NP-40 lysis buffer, combine 30 mL of 5 M NaCl, 100 mL of 10% NP-40, 50 mL of 1 M Tris (pH 8.0), and 820 mL of H2O. Store at 4˚C. Triton X-100 can be used with similar results. Useful variations include lowering the detergent concentration, raising the salt concentration, or switching to other detergents such as saponin, digitonin, or CHAPS. PBS (A)
Reagent NaCl KCl KH2PO4 NaH2PO4
Amount to add for 1 L
Final concentration
8.0 g 0.2 g 0.2 g 1.14 g
137.0 mM 2.7 mM 1.5 mM 8.0 mM
Adjust pH to 7.2 with HCl (4–6 droplets of 6 M HCl).
SDS-PAGE Running Buffer
25 mM Tris-Cl 192 mM glycine 0.1% SDS SDS-PAGE Sample Buffer (4×)
40% glycerol 200 mM Tris-Cl (pH 6.8) 8% SDS 4% β-mercaptoethanol 0.02% bromophenol blue Western Transfer Buffer
25 mM Tris-Cl 192 mM glycine 20% (v/v) methanol Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080291
787
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
G.P. McStay and D.R. Green
ACKNOWLEDGMENTS
Supported by grants from the National Institutes of Health. REFERENCES Inoue S, Browne G, Melino G, Cohen GM. 2009. Ordering of caspases in cells undergoing apoptosis by the intrinsic pathway. Cell Death Differ 16: 1053–1061.
788
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080291
Detection of Caspase Activity Using Antibody-Based Techniques Gavin P. McStay and Douglas R. Green Cold Spring Harb Protoc; doi: 10.1101/pdb.prot080291 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) Immunoblotting (24 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 UNIV OF CALIF-SF on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Protocol
Detection of Caspase Activity Using Antibody-Based Techniques 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
A number of antibodies have been generated that recognize caspases from mammalian model organisms. These include antibodies that recognize specific caspase pro-forms and others that bind caspase cleavage fragments. These antibodies are excellent reagents for identifying which executioner caspases have been activated following application or induction of a specific apoptotic stimulus. This approach is more difficult to use with initiator caspases, however, because cleavage does not necessarily correlate with caspase activation. In this protocol, cultured cells are treated with a proapoptotic stimulus, and then protein lysates are prepared from the treated cells. The proteins are then separated by gel electrophoresis and transferred to a suitable membrane. The fragment-specific antibodies that recognize executioner caspases are used in a western analysis to determine the extent of activation and to aid in identifying which caspases have been activated.
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. RECIPES: 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
Antibody to caspase, recognizing either full-length protein or a cleavage fragment Antibody recognizing primary antibody, conjugated to horseradish peroxidase Bradford protein assay materials (see Step 6) Cell line to be treated with an apoptotic stimulus Dry ice–methanol bath (optional; see Step 4) Enhanced chemiluminescent reagent (Thermo Scientific) Homogenization buffer for caspase activation (optional; see Step 4) Nonfat dry milk (5%) in TBS-T NP-40 lysis buffer Add protease inhibitors (e.g., complete protease inhibitor cocktail tablets [Roche]) to the NP-40 lysis buffer according to the manufacturer’s instructions.
Pan-caspase inhibitor (e.g., methylated zVAD-FMK) PBS (A) 3
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080291
783
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
G.P. McStay and D.R. Green
Proapoptotic stimulus The apoptotic stimulus can be a reagent like etoposide, a ligand like anti-Fas antibody, or a physical perturbation like UV radiation.
SDS–PAGE running buffer SDS–PAGE sample buffer (4×) TBS-T buffer (e.g., Abcam or Cell Signaling Technology) Trypsin–EDTA (e.g., trypsin–EDTA in PBS [Life Technologies]) Western transfer buffer Equipment
Autoradiography film, film cassette, and film processor Centrifuge (benchtop) for 15-mL conical tubes Heating block set to 95˚C Microcentrifuge at 4˚C Nitrocellulose membrane SDS–PAGE equipment Western transfer apparatus METHOD A flowchart of this protocol is shown in Figure 1.
1. Treat cells with a proapoptotic stimulus. Inclusion of negative controls is important at this stage. Include two types of negative controls: (1) cells not treated with the proapoptotic stimulus and (2) cells pretreated with a pan-caspase inhibitor that is able to enter cells (e.g., methylated zVAD-FMK). In the latter control, incubate cells and the pan-caspase inhibitor for 1 h before treatment with proapoptotic stimulus. The concentration of the inhibitor depends on which inhibitor is included (e.g., use 100 µM methylated zVAD-FMK or 20 µM QVD-OPH).
Harvest treated cells and wash with PBS Ensure all floating/apoptotic cells are harvested Lyse cells in NP-40 lysis buffer with protease inhibitors
Centrifuge, keep supernatant 15,000g, 10 min, 4°C Determine protein concentration Depends on laboratory preferences Add SDS-PAGE sample buffer to sample 95°C, 10 min Run 20 μg of the heated sample on SDS-PAGE Conditions depend on laboratory preferences Western transfer Conditions depend on laboratory preferences Immunoblot with caspase-specific antibody and secondary antibody Detect protein: antibody complexes using chemiluminescence
784
FIGURE 1. Detection of caspase activity using antibody-based techniques. This protocol requires 1–1.5 d to complete. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080291
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Detecting Caspase Activity with Antibodies
2. Harvest treated cells by collecting the medium (containing detached apoptotic cells) and releasing adherent cells from the dish with trypsin–EDTA solution. Combine all of the cells in a centrifuge tube. Collect the cells by centrifugation at 400g for 5 min at room temperature. 3. Wash the cells in PBS and collect them by centrifugation as in Step 2. 4. Discard the supernatant. Add 100 µL of NP-40 lysis buffer with protease inhibitors per 5 × 105 cells. Lyse the cells by pipetting the cell pellet up and down until no clumps remain. Incubate the cells on ice for 10 min. Alternatively, cytosolic extracts can be prepared by resuspending cell pellets in homogenization buffer for caspase activation, followed by three freeze–thaw cycles in a dry ice-methanol bath.
5. Centrifuge the lysate at 15,000g for 10 min at 4˚C.
6. Determine the protein concentration of the supernatant using the Bradford assay or another suitable method. 7. Add 4× SDS–PAGE sample buffer to an appropriate volume of cell lysate containing 20 µg of protein. Heat at 95˚C for 10 min.
8. Run the heated lysate on a 12% SDS–PAGE gel at 200 V until the gel front dye reaches the bottom of the gel. 9. Transfer the separated proteins onto nitrocellulose using standard western transfer techniques and western transfer buffer. 10. Block the membrane with 5% nonfat dry milk in TBS-T for 30 min at room temperature. Rock the membrane gently during blocking. 11. Wash the membrane in TBS-T. Incubate the membrane in primary antibody (dilution depends on the antibody being used) in 5% nonfat dry milk in TBS-T solution for 2 h, rocking at room temperature (or overnight at 4˚C). 12. Remove the antibody solution. Wash the membrane three times for 5 min each by rocking in TBS-T at room temperature.
13. Incubate the membrane in secondary antibody (1:10,000 dilution) in 5% nonfat dry milk in TBST solution for 2 h with rocking at room temperature. 14. Remove the antibody solution. Wash the membrane three times for 5 min each by rocking in TBS-T at room temperature. 15. Expose the membrane to enhanced chemiluminescent reagent for 1 min. 16. Cover the membrane with plastic and expose to X-ray film for different amounts of time (seconds to minutes) to detect bands corresponding to full length and cleaved caspases. Other methods for chemiluminescence capture are suitable, such as a gel documentation system that has chemiluminescent detection capability. The exposed western blot should show smaller protein bands corresponding to the cleaved form of the caspases (15 kDa–20 kDa for executioner caspases and 40 kDa for initiator caspases). If these are true caspase cleavage products, then the bands should be absent from both the untreated cells and the cells pretreated with pan-caspase inhibitor before treatment with proapoptotic stimulus (Fig. 2). See Troubleshooting.
TROUBLESHOOTING Problem (Step 16): No cleaved caspases are seen in apoptotic extracts. Solution: Some caspases (such as caspase-7 and caspase-9) once cleaved become susceptible to deg-
radation in an ubiquitin-dependent manner. To improve the detection of these cleavage products, take samples at early time points (several hours instead of overnight treatment). It is also possible to use a proteasome inhibitor, such as MG132, to prevent degradation. There are also some Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080291
785
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
G.P. McStay and D.R. Green
37 Pro-caspase-3 25
FIGURE 2. Caspase-3 western blot of Jurkat cells preincubated with 100 µM zVAD-FMK for 10 min before the induction of apoptosis overnight. The untreated samples show caspase-3 as full-length procaspase at 30 kDa. In the apoptotic sample there is less full-length caspase-3 and a 15 kDa band appears, representing cleaved caspase-3 fragments. In cells preincubated with zVAD-FMK before treatment with the proapoptotic stimulus, only full-length caspase-3 is seen, indicating that cleavage was caspase dependent.
Cleaved caspase-3
15 –
+
–
+
zVAD-FMK
Ap
Un
op
tre
to
at
tic
ed
circumstances in which caspases remain uncleaved when apoptosis is triggered. This is especially true for initiator caspases (see Discussion). DISCUSSION
Detection of caspase cleavage products by western blotting is a straightforward method for implicating a specific caspase in a particular death pathway. However, this approach is crude, because it does not take into account the feedback that occurs in apoptotic pathways or the mechanisms of activation of initiator caspases. Monitoring cleavage events of executioner caspases provides a direct read-out of activation. The disappearance of the full-length protein, coupled with the appearance of the cleaved fragments containing the large and small catalytic subunits, indicates that one or more executioner caspases have been activated (Fig. 3). The same methodology does not yield comparable results with initiator caspases. Although cleavage of an initiator caspase is sometimes necessary for its activity, it is not sufficient to activate it. Thus, simply monitoring the cleavage of an initiator caspase does not tell us whether the caspase dimerized—and was therefore truly activated—before cleavage. In general, however, it is true that if an initiator caspase is not cleaved, then it is unactivated (Fig. 3). Cleaved initiator caspases typically function as substrates of executioner caspases as part of the controlled demolition of a cell (Inoue et al. 2009).
RELATED TECHNIQUES
Antibodies that recognize specific cleaved fragments of caspases are available, and these are commonly used in ELISAs or immunohistochemical staining of tissue to detect active caspases.
Initiator
1
2
Executioner
3
4
5
6
Full length Cleaved
Active
786
?
?
?
No
Yes
Yes
FIGURE 3. Schematic representation of a caspase immunoblot depicting problems of interpreting activity based on caspase cleavage patterns. Initiator caspases in lanes 1, 2, and 3, whether uncleaved, cleaved, or a mixture, do not reveal whether the caspase was activated. Executioner caspases in lanes 4, 5, and 6 show that uncleaved executioner caspases are not active and cleaved executioner caspases are active.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080291
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Detecting Caspase Activity with Antibodies
RECIPES Homogenization Buffer for Caspase Activation
10 mM HEPES (pH 7.0) 5 mM MgCl2 0.67 mM DTT 1 protease inhibitor cocktail tablet (Roche) for the appropriate volume of homogenization buffer The homogenization buffer without DTT or protease inhibitors can be stored at –4˚C for up to 6 mo. Once DTT and protease inhibitors are added, the homogenization buffer should be stored at –20˚C in 10-mL aliquots; it will last for up to 1 yr under these conditions. NP-40 Lysis Buffer
NaCl (150 mM) NP-40 (1.0%) Tris-Cl (50 mM, pH 8.0) For 1 L of NP-40 lysis buffer, combine 30 mL of 5 M NaCl, 100 mL of 10% NP-40, 50 mL of 1 M Tris (pH 8.0), and 820 mL of H2O. Store at 4˚C. Triton X-100 can be used with similar results. Useful variations include lowering the detergent concentration, raising the salt concentration, or switching to other detergents such as saponin, digitonin, or CHAPS. PBS (A)
Reagent NaCl KCl KH2PO4 NaH2PO4
Amount to add for 1 L
Final concentration
8.0 g 0.2 g 0.2 g 1.14 g
137.0 mM 2.7 mM 1.5 mM 8.0 mM
Adjust pH to 7.2 with HCl (4–6 droplets of 6 M HCl).
SDS-PAGE Running Buffer
25 mM Tris-Cl 192 mM glycine 0.1% SDS SDS-PAGE Sample Buffer (4×)
40% glycerol 200 mM Tris-Cl (pH 6.8) 8% SDS 4% β-mercaptoethanol 0.02% bromophenol blue Western Transfer Buffer
25 mM Tris-Cl 192 mM glycine 20% (v/v) methanol Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080291
787
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
G.P. McStay and D.R. Green
ACKNOWLEDGMENTS
Supported by grants from the National Institutes of Health. REFERENCES Inoue S, Browne G, Melino G, Cohen GM. 2009. Ordering of caspases in cells undergoing apoptosis by the intrinsic pathway. Cell Death Differ 16: 1053–1061.
788
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080291
