CHAPTER NINETEEN
Western Blotting using Chemiluminescent Substrates Alice Alegria-Schaffer1 Thermo Fisher Scientific, Rockford, IL, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Duration 4.2 Preparation 5. Step 1: Protein Transfer to a Membrane 5.1 Overview 5.2 Duration 5.3 Tip 5.4 Tip 6. Step 2: Western Blot Detection using a Chemiluminescent Substrate 6.1 Overview 6.2 Duration 6.3 Tip 6.4 Tip 6.5 Tip References Source References
252 252 252 253 253 253 253 254 254 254 255 256 256 256 256 257 258 258 258 259
Abstract Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture (Towbin et al., 1979). The technique enables indirect detection of protein samples immobilized on a nitrocellulose or polyvinylidene fluoride (PVDF) membrane.
Methods in Enzymology, Volume 541 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420119-4.00019-7
#
2014 Elsevier Inc. All rights reserved.
251
252
Alice Alegria-Schaffer
1. THEORY To detect the target protein, a primary antibody (polyclonal or monoclonal) against the target antigen is applied to the membrane as a probe. The membrane is washed and incubated with a horseradish peroxidase (HRP)conjugated secondary antibody that is reactive toward the primary antibody. The membrane is washed again and incubated with an appropriate luminolbased chemiluminescent substrate. In the presence of HRP and a peroxide buffer, luminol oxidizes and forms an excited state product that emits light as it decays to the ground state. Light emission occurs only during the enzymesubstrate reaction and, therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases. The signal is evaluated with X-ray film or imaging instrumentation.
2. EQUIPMENT Polyacrylamide gel electrophoresis equipment Gel transfer unit Platform rocker Autoradiography film and film developer, or a cooled CCD camera Nitrocellulose or PVDF membrane Blotting paper (e.g., Whatman 3MM chromatography paper) Small plastic containers
3. MATERIALS Target-specific primary antibody Horseradish peroxidase (HRP)-conjugated secondary antibody HRP chemiluminescent substrate Nonfat dry milk Glycine Tris base Methanol Sodium phosphate monobasic (NaH2PO4) Sodium phosphate dibasic (Na2HPO4) Sodium chloride (NaCl) Tween-20 Pierce Reversible Protein Stain (Thermo Scientific) (optional)
253
Western Blotting using Chemiluminescent Substrates
3.1. Solutions & buffers Step 1 Tris–glycine transfer buffer Component
Final concentration
Amount
Tris base
25 mM
3g
Glycine
192 mM
14.4 g
20%
200 ml
Add water to 800 ml Methanol
Step 2 Phosphate-buffered saline with tween-20 (PBS-T) Component
Final concentration
Stock
Amount
Monobasic sodium phosphate
100 mM
0.2 M
140 ml
0.2 M
360 ml
Dibasic sodium phosphate heptahydrate Tween-20
0.05%
10%
5 ml
Sodium chloride
150 mM
5M
30 ml
Add water to 1 l
Blocking buffer Component
Final concentration
PBS-T
Amount
1l
Nonfat dry milk
5%
50 g
4. PROTOCOL 4.1. Duration Preparation
About 1 day
Protocol
About 4 h to overnight
4.2. Preparation Prepare sample and separate proteins on a polyacrylamide gel by SDS-PAGE (See One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDSPAGE)). Prepare buffers.
254
Alice Alegria-Schaffer
Figure 19.1 Flowchart of the complete protocol, including preparation.
See Fig. 19.1 for the flowchart of the complete protocol.
5. STEP 1: PROTEIN TRANSFER TO A MEMBRANE 5.1. Overview Proteins are transferred from the SDS-PAGE gel to a PVDF or nitrocellulose membrane.
5.2. Duration 1–2 h 1.1 Remove the gel from the electrophoresis unit and equilibrate in cold Transfer Buffer for 10–30 min with gentle shaking. Incubation time is based on a 1.5 mm thick gel. Thinner gels will require less incubation time. See Video 19.1, http://dx.doi.org/10.1016/B978-0-12420119-4.00019-7. 1.2 Cut the membrane to the dimensions of the gel. Cut a notch in the membrane corner to correspond to a corner of the gel. 1.3 Place the membrane in a container with cold Transfer Buffer until ready to use. For PVDF membranes, wet briefly in methanol, rinse several times with dH2O, and then place in Transfer Buffer. 1.4 Wet the absorbent blotting paper and transfer pads in Transfer Buffer. 1.5 Use the following component order to form the transfer stack (Fig. 19.2 and Video 19.2): http://dx.doi.org/10.1016/B978-0-12-420119-4. 00019-7 • Anode (red) • Pad (for wet transfer) • Blotting paper (2–3 sheets) • Gel • Membrane
Western Blotting using Chemiluminescent Substrates
255
Figure 19.2 Diagram of the transfer stack for Western transfer.
1.6 1.7 1.8 1.9
• Blotting paper (2–3 sheets) • Pad (for wet transfer) • Cathode (black) Ensure that there are no bubbles between the gel and the membrane that would prevent proper transfer by using a gel roller after placing the membrane. Connect the leads and perform transfer for 45–90 min at 0.8 mA cm 2 of gel. See Video 19.4, http://dx.doi.org/10.1016/B978-0-12420119-4.00019-7. When the transfer is complete, disconnect leads and disassemble the transfer stack to remove the membrane. Keep membrane moist until ready to use. To evaluate transfer efficiency, stain the membrane with a reversible protein stain.
5.3. Tip Use clean and dry scissors to cut membrane to the size of the gel. Any small tear may result in a larger tear. Always wear gloves while handling membranes because oils from fingers may prevent proper wetting and proteins from hands may bind to the membrane causing background.
256
Alice Alegria-Schaffer
Figure 19.3 Flowchart of Step 1.
5.4. Tip Transfer time and efficiency will vary depending upon polyacrylamide concentration, gel thickness, the presence of SDS or organic solvents, pH and ionic strength of the transfer buffer, and the molecular weight of the protein. Empirically determine optimal transfer conditions. See Fig. 19.3 for the flowchart of Step 1.
6. STEP 2: WESTERN BLOT DETECTION USING A CHEMILUMINESCENT SUBSTRATE 6.1. Overview Proteins immobilized on a PVDF or nitrocellulose membrane are indirectly detected via a target-specific primary antibody and an HRP-labeled secondary antibody. A chemiluminescent enzyme substrate is used to visualize the target.
6.2. Duration 3 h – overnight 2.1 Remove the membrane and block the nonspecific binding sites with blocking buffer for 20–60 min at room temperature with shaking.
257
Western Blotting using Chemiluminescent Substrates
Table 19.1 Primary and secondary antibody concentrations to use with thermo scientific chemiluminescent substrates West Duraa West Femtoa Substrate ECL West Picoa
Primary antibody concentration (mg ml 1)
0.2–10
0.2–1.0
0.02–1.0
0.01–0.2
Secondary antibody concentration (ng ml 1)
67–1000
10–50
4–20
2–10
a
Part of the thermo scientific supersignal product family.
2.2 Incubate the blot with the primary antibody solution (see Table 19.1) diluted in blocking buffer with rocking for 1 h. If desired, incubate the blot overnight at 2–8 C. 2.3 Wash the membrane three times for 5 min each with PBS-T. 2.4 Incubate the blot with the HRP-conjugated secondary antibody (see Table 19.1) containing 10% blocking solution for 1 h with rocking at room temperature. 2.5 Wash the membrane five times for 5 min each in PBS-T to remove any nonbound conjugate. 2.6 Prepare the chemiluminescent substrate according to the manufacturer’s instructions. Use a sufficient volume to ensure that the blot is completely wetted with the substrate and the blot does not become dry (0.1 ml cm 2). 2.7 Incubate the blot with the substrate according to the manufacturer’s instructions. For example, incubate for 1 min when using ECL or 5 min when using SuperSignal Substrates. 2.8 Remove the blot from the substrate and place it in a plastic membrane protector. See Video 19.3, http://dx.doi.org/10.1016/B978-0-12420119-4.00019-7. A plastic sheet protector works well, although plastic wrap may also be used. Remove all air bubbles between the blot and the surface of the membrane protector. 2.9 Image the blot using film or a cooled CCD camera.
6.3. Tip Other proteinaceous buffers for blocking may be used, such as 3% fraction V, crystalline-grade bovine serum albumin.
258
Alice Alegria-Schaffer
Figure 19.4 Flowchart of Step 2.
6.4. Tip Use the primary and secondary antibodies at the concentrations recommended by the manufacturer of the chemiluminescent substrate you are using. Antibody concentration might require optimization.
6.5. Tip To minimize background, it is crucial to thoroughly wash the membrane after incubation with the enzyme conjugate. See Fig. 19.4 for the flowchart of Step 2.
REFERENCES Referenced Literature Towbin, H., Staehelin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America, 76, 4350–4354.
Western Blotting using Chemiluminescent Substrates
259
SOURCE REFERENCES Alegria-Schaffer, A., Lodge, A., & Vattem, K. (2009). Performing and optimizing Western blots with an emphasis on chemiluminescent detection. Methods in Enzymology, 463, 573–599.
Referenced Protocols in Methods Navigator One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE).
Western Blotting using Chemiluminescent Substrates Alice Alegria-Schaffer1 Thermo Fisher Scientific, Rockford, IL, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Duration 4.2 Preparation 5. Step 1: Protein Transfer to a Membrane 5.1 Overview 5.2 Duration 5.3 Tip 5.4 Tip 6. Step 2: Western Blot Detection using a Chemiluminescent Substrate 6.1 Overview 6.2 Duration 6.3 Tip 6.4 Tip 6.5 Tip References Source References
252 252 252 253 253 253 253 254 254 254 255 256 256 256 256 257 258 258 258 259
Abstract Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture (Towbin et al., 1979). The technique enables indirect detection of protein samples immobilized on a nitrocellulose or polyvinylidene fluoride (PVDF) membrane.
Methods in Enzymology, Volume 541 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420119-4.00019-7
#
2014 Elsevier Inc. All rights reserved.
251
252
Alice Alegria-Schaffer
1. THEORY To detect the target protein, a primary antibody (polyclonal or monoclonal) against the target antigen is applied to the membrane as a probe. The membrane is washed and incubated with a horseradish peroxidase (HRP)conjugated secondary antibody that is reactive toward the primary antibody. The membrane is washed again and incubated with an appropriate luminolbased chemiluminescent substrate. In the presence of HRP and a peroxide buffer, luminol oxidizes and forms an excited state product that emits light as it decays to the ground state. Light emission occurs only during the enzymesubstrate reaction and, therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases. The signal is evaluated with X-ray film or imaging instrumentation.
2. EQUIPMENT Polyacrylamide gel electrophoresis equipment Gel transfer unit Platform rocker Autoradiography film and film developer, or a cooled CCD camera Nitrocellulose or PVDF membrane Blotting paper (e.g., Whatman 3MM chromatography paper) Small plastic containers
3. MATERIALS Target-specific primary antibody Horseradish peroxidase (HRP)-conjugated secondary antibody HRP chemiluminescent substrate Nonfat dry milk Glycine Tris base Methanol Sodium phosphate monobasic (NaH2PO4) Sodium phosphate dibasic (Na2HPO4) Sodium chloride (NaCl) Tween-20 Pierce Reversible Protein Stain (Thermo Scientific) (optional)
253
Western Blotting using Chemiluminescent Substrates
3.1. Solutions & buffers Step 1 Tris–glycine transfer buffer Component
Final concentration
Amount
Tris base
25 mM
3g
Glycine
192 mM
14.4 g
20%
200 ml
Add water to 800 ml Methanol
Step 2 Phosphate-buffered saline with tween-20 (PBS-T) Component
Final concentration
Stock
Amount
Monobasic sodium phosphate
100 mM
0.2 M
140 ml
0.2 M
360 ml
Dibasic sodium phosphate heptahydrate Tween-20
0.05%
10%
5 ml
Sodium chloride
150 mM
5M
30 ml
Add water to 1 l
Blocking buffer Component
Final concentration
PBS-T
Amount
1l
Nonfat dry milk
5%
50 g
4. PROTOCOL 4.1. Duration Preparation
About 1 day
Protocol
About 4 h to overnight
4.2. Preparation Prepare sample and separate proteins on a polyacrylamide gel by SDS-PAGE (See One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDSPAGE)). Prepare buffers.
254
Alice Alegria-Schaffer
Figure 19.1 Flowchart of the complete protocol, including preparation.
See Fig. 19.1 for the flowchart of the complete protocol.
5. STEP 1: PROTEIN TRANSFER TO A MEMBRANE 5.1. Overview Proteins are transferred from the SDS-PAGE gel to a PVDF or nitrocellulose membrane.
5.2. Duration 1–2 h 1.1 Remove the gel from the electrophoresis unit and equilibrate in cold Transfer Buffer for 10–30 min with gentle shaking. Incubation time is based on a 1.5 mm thick gel. Thinner gels will require less incubation time. See Video 19.1, http://dx.doi.org/10.1016/B978-0-12420119-4.00019-7. 1.2 Cut the membrane to the dimensions of the gel. Cut a notch in the membrane corner to correspond to a corner of the gel. 1.3 Place the membrane in a container with cold Transfer Buffer until ready to use. For PVDF membranes, wet briefly in methanol, rinse several times with dH2O, and then place in Transfer Buffer. 1.4 Wet the absorbent blotting paper and transfer pads in Transfer Buffer. 1.5 Use the following component order to form the transfer stack (Fig. 19.2 and Video 19.2): http://dx.doi.org/10.1016/B978-0-12-420119-4. 00019-7 • Anode (red) • Pad (for wet transfer) • Blotting paper (2–3 sheets) • Gel • Membrane
Western Blotting using Chemiluminescent Substrates
255
Figure 19.2 Diagram of the transfer stack for Western transfer.
1.6 1.7 1.8 1.9
• Blotting paper (2–3 sheets) • Pad (for wet transfer) • Cathode (black) Ensure that there are no bubbles between the gel and the membrane that would prevent proper transfer by using a gel roller after placing the membrane. Connect the leads and perform transfer for 45–90 min at 0.8 mA cm 2 of gel. See Video 19.4, http://dx.doi.org/10.1016/B978-0-12420119-4.00019-7. When the transfer is complete, disconnect leads and disassemble the transfer stack to remove the membrane. Keep membrane moist until ready to use. To evaluate transfer efficiency, stain the membrane with a reversible protein stain.
5.3. Tip Use clean and dry scissors to cut membrane to the size of the gel. Any small tear may result in a larger tear. Always wear gloves while handling membranes because oils from fingers may prevent proper wetting and proteins from hands may bind to the membrane causing background.
256
Alice Alegria-Schaffer
Figure 19.3 Flowchart of Step 1.
5.4. Tip Transfer time and efficiency will vary depending upon polyacrylamide concentration, gel thickness, the presence of SDS or organic solvents, pH and ionic strength of the transfer buffer, and the molecular weight of the protein. Empirically determine optimal transfer conditions. See Fig. 19.3 for the flowchart of Step 1.
6. STEP 2: WESTERN BLOT DETECTION USING A CHEMILUMINESCENT SUBSTRATE 6.1. Overview Proteins immobilized on a PVDF or nitrocellulose membrane are indirectly detected via a target-specific primary antibody and an HRP-labeled secondary antibody. A chemiluminescent enzyme substrate is used to visualize the target.
6.2. Duration 3 h – overnight 2.1 Remove the membrane and block the nonspecific binding sites with blocking buffer for 20–60 min at room temperature with shaking.
257
Western Blotting using Chemiluminescent Substrates
Table 19.1 Primary and secondary antibody concentrations to use with thermo scientific chemiluminescent substrates West Duraa West Femtoa Substrate ECL West Picoa
Primary antibody concentration (mg ml 1)
0.2–10
0.2–1.0
0.02–1.0
0.01–0.2
Secondary antibody concentration (ng ml 1)
67–1000
10–50
4–20
2–10
a
Part of the thermo scientific supersignal product family.
2.2 Incubate the blot with the primary antibody solution (see Table 19.1) diluted in blocking buffer with rocking for 1 h. If desired, incubate the blot overnight at 2–8 C. 2.3 Wash the membrane three times for 5 min each with PBS-T. 2.4 Incubate the blot with the HRP-conjugated secondary antibody (see Table 19.1) containing 10% blocking solution for 1 h with rocking at room temperature. 2.5 Wash the membrane five times for 5 min each in PBS-T to remove any nonbound conjugate. 2.6 Prepare the chemiluminescent substrate according to the manufacturer’s instructions. Use a sufficient volume to ensure that the blot is completely wetted with the substrate and the blot does not become dry (0.1 ml cm 2). 2.7 Incubate the blot with the substrate according to the manufacturer’s instructions. For example, incubate for 1 min when using ECL or 5 min when using SuperSignal Substrates. 2.8 Remove the blot from the substrate and place it in a plastic membrane protector. See Video 19.3, http://dx.doi.org/10.1016/B978-0-12420119-4.00019-7. A plastic sheet protector works well, although plastic wrap may also be used. Remove all air bubbles between the blot and the surface of the membrane protector. 2.9 Image the blot using film or a cooled CCD camera.
6.3. Tip Other proteinaceous buffers for blocking may be used, such as 3% fraction V, crystalline-grade bovine serum albumin.
258
Alice Alegria-Schaffer
Figure 19.4 Flowchart of Step 2.
6.4. Tip Use the primary and secondary antibodies at the concentrations recommended by the manufacturer of the chemiluminescent substrate you are using. Antibody concentration might require optimization.
6.5. Tip To minimize background, it is crucial to thoroughly wash the membrane after incubation with the enzyme conjugate. See Fig. 19.4 for the flowchart of Step 2.
REFERENCES Referenced Literature Towbin, H., Staehelin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America, 76, 4350–4354.
Western Blotting using Chemiluminescent Substrates
259
SOURCE REFERENCES Alegria-Schaffer, A., Lodge, A., & Vattem, K. (2009). Performing and optimizing Western blots with an emphasis on chemiluminescent detection. Methods in Enzymology, 463, 573–599.
Referenced Protocols in Methods Navigator One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE).
