CHAPTER TWELVE
One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE) Julie L. Brunelle*,†, Rachel Green*,†,1
*Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA † Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, 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 Casting an SDS-PAGE Gel: Resolving Gel 5.1 Overview 5.2 Duration 5.3 Tip 5.4 Tip 5.5 Caution 6. Step 2 Casting an SDS-PAGE Gel: Stacking Gel 6.1 Overview 6.2 Duration 7. Step 3 Running an SDS-PAGE Gel 7.1 Overview 7.2 Duration 7.3 Tip References
152 152 153 153 154 154 154 154 154 154 155 155 155 155 155 156 157 157 157 159 159
Abstract This protocol describes a denaturing polyacrylamide gel system utilizing sodium dodecyl sulfate (SDS) to separate protein molecules based on size as first described by Laemmli (1970). SDS-PAGE can be used to monitor protein purifications, check the purity of samples, and to estimate molecular weights for unknown proteins.
Methods in Enzymology, Volume 541 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420119-4.00012-4
#
2014 Elsevier Inc. All rights reserved.
151
152
Julie L. Brunelle and Rachel Green
1. THEORY Polyacrylamide gel electrophoresis is useful for separating molecules by size and charge and there are many different systems depending on the sample and downstream applications. SDS-PAGE is a very useful tool to separate protein molecules by size. SDS is a detergent that denatures secondary and nondisulfide-linked tertiary structures and coats them with a negative charge that correlates with their length, allowing molecular weights to be estimated. Mobility through the gel can be affected by the state of the protein (e.g., phosphorylation and presence of multimeric molecules). The Laemmli SDS-PAGE system is a discontinuous gel with an upper stacking gel and lower resolving gel that have different pH values and polyacrylamide concentrations. The upper stacking gel has a lower percentage of polyacrylamide allowing proteins to move through quickly and ‘stack’ into a tight band before entering into the higher percentage polyacrylamide resolving gel for separation. The percentages of polyacrylamide can be optimized for the size range of molecules present in the sample. Gradient gels can be prepared allowing a greater range of separation in a single gel if both large and small proteins need to be resolved simultaneously. Small proteins will move through the resolving gel more quickly than large proteins. The protocol below is for mini gels, but can be scaled up for larger gel plates easily and directly. The method can be simplified greatly by using commercially available precast gels.
2. EQUIPMENT PAGE gel casting stand PAGE gel rig Glass plates Spacers Gel combs Power supply Pipet-aid Glass or disposable pipettes (5 ml and 10 ml) 10-ml syringe Needle (18–25 gauge) Micropipettors
153
One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE)
Micropipettor tips Gel loading tips 15-ml conical polypropylene tubes
3. MATERIALS Tris base Sodium dodecyl sulfate (SDS) Glycerol b-mercaptoethanol Bromophenol blue Glycine Hydrochloric acid (HCl) 40% acrylamide/bisacrylamide solution mix (19:1) Ammonium persulfate (APS) Tetramethylethylenediamine (TEMED) MilliQ H2O Protein molecular weight marker
3.1. Solutions & buffers Step 3 SDS-PAGE sample buffer Component
Final concentration
Stock
Amount
Tris–HCl, pH 6.8
120 mM
1M
0.6 ml
SDS
4%
10%
2 ml
Glycerol
20%
100%
1 ml
b-mercaptoethanol
750 mM
14.3 M
0.267 ml
Bromophenol blue
0.05%
Add water to 5 ml. Store in 0.5 ml aliquots at
0.0025 g 20 C
SDS-PAGE running buffer Component
Final concentration
Tris base
25 mM
3.025 g
Glycine
192 mM
14.4 g
SDS
0.1%
Add water to 1 l
Stock
10%
Amount
10 ml
154
Julie L. Brunelle and Rachel Green
4. PROTOCOL 4.1. Duration Preparation
5 min
Protocol
3–4 h
4.2. Preparation Clean the glass plates, spacers, and combs. Assemble the gel-casting sandwich and insert the comb to mark a line for the resolving gel height 1 cm below the bottom of the comb. See Fig. 12.1 for the flowchart of the complete protocol.
5. STEP 1 CASTING AN SDS-PAGE GEL: RESOLVING GEL 5.1. Overview Prepare and pour the lower 12% resolving gel.
5.2. Duration 1h 1.1 Place gel-casting sandwich into the casting stand. See Video 12.1, http://dx.doi.org/10.1016/B978-0-12-420119-4.00012-4. 1.2 Prepare the 12% resolving SDS gel solution:
Figure 12.1 Flowchart of the complete protocol, including preparation.
155
One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE)
Component
Final concentration
Stock
Amount
Acrylamide/bisacrylamide (19:1)
12%
40%
3 ml
SDS
0.1%
10%
0.1 ml
Tris–HCl, pH 8.8
375 mM
1.5 M
2.5 ml
Water
4.4 ml
1.3 Add 50 ml 10% APS and 5 ml TEMED; mix gently and quickly. See Video 12.2, http://dx.doi.org/10.1016/B978-0-12-420119-4.00012-4. 1.4 Pipette the resolving gel solution into the gel casting sandwich up to the line marked on the plate. 1.5 Overlay the gel with a layer of water-saturated butanol to smooth the top of the gel surface and aid in polymerization. 1.6 Allow the resolving gel to polymerize for 1 h. 1.7 Rinse the resolving gel with MilliQ water and remove any water droplets with a kimwipe. See Video 12.3, http://dx.doi.org/10.1016/ B978-0-12-420119-4.00012-4.
5.3. Tip APS and TEMED catalyze the polymerization of acrylamide. Once these are added, act quickly to pour the gel before it polymerizes.
5.4. Tip The percentage of acrylamide in the resolving gel can be adjusted to achieve the desired separation of proteins. 12% is used in this example and refers to the total concentration of acrylamide and bisacrylamide in the solution; 4–17% is the useful range for SDSPAGE.
5.5. Caution Acrylamide is a dangerous neurotoxin readily absorbed through the skin. Use gloves at all times. See Fig. 12.2 for the flowchart of Step 1.
6. STEP 2 CASTING AN SDS-PAGE GEL: STACKING GEL 6.1. Overview Prepare and pour the upper 5% stacking gel.
156
Julie L. Brunelle and Rachel Green
Figure 12.2 Flowchart of Step 1.
6.2. Duration 45 min 2.1 Prepare 5% stacking SDS gel solution: Component
Final concentration
Stock
Amount
Acrylamide/bisacrylamide (19:1)
5%
40%
0.623 ml
SDS
0.1%
10%
0.05 ml
Tris–HCl, pH 6.8
125 mM
1M
0.63 ml
Water
3.697 ml
2.2 Add 50 ml 10% APS and 10 ml TEMED; mix gently and quickly. See Video 12.4, http://dx.doi.org/10.1016/B978-0-12-420119-4.00012-4. 2.3 Pipette the stacking gel solution on top of the resolving gel in the gelcasting sandwich, filling to the top of the plate. 2.4 Insert the comb carefully to avoid trapping air bubbles; apply additional gel solution to fill in any low areas. 2.5 Allow the stacking gel to polymerize for 30–45 min.
One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE)
157
Figure 12.3 Flowchart of Step 2.
See Fig. 12.3 for the flowchart of Step 2.
7. STEP 3 RUNNING AN SDS-PAGE GEL 7.1. Overview Assemble gel rig and load samples to perform separation by applying electric current. See Video 12.5, http://dx.doi.org/10.1016/B978-0-12-420119-4. 00012-4.
7.2. Duration 1.5 h 3.1 Place the polymerized SDS-PAGE gel sandwich into the PAGE gel rig. 3.2 Add SDS gel running buffer to the upper and lower chambers. Check for bubbles along the bottom surface of the gel and remove by flushing with running buffer. 3.3 Remove the comb and rinse the wells with running buffer using a 10-ml syringe with a needle to flush away any unpolymerized acrylamide solution. 3.4 Dilute protein sample(s) at least 1:2 with SDS-PAGE sample buffer and heat at 95 C for 1–3 min.
158
3.5
Julie L. Brunelle and Rachel Green
Load molecular weight marker and prepared protein samples in the wells, using a micropipettor with gel loading tips. See Video 12.6, http://dx.doi.org/10.1016/B978-0-12-420119-4.00012-4. 3.6 Attach electrical leads to the gel rig and to the power supply matching the red and black. 3.7 Turn on the power supply to a constant 150–200 V for 35 min to 1 h. 3.8 When the bromophenol blue has run to the bottom of the gel, turn off the power supply and disconnect the leads. See Video 12.7, http://dx.doi.org/10.1016/B978-0-12-420119-4.00012-4. 3.9 Discard the running buffer and rinse the gel sandwich with water. 3.10 Carefully separate the glass plates to access the gel. 3.11 Proceed with downstream applications [e.g., staining (see Coomassie Blue Staining or Silver Staining of SDS-polyacrylamide Gel) or Western transfer (see Western Blotting using Chemiluminescent Substrates)].
Figure 12.4 Flowchart of Step 3.
One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE)
7.3. Tip 5–20 mg of protein can be detected by Coomassie blue staining. See Fig. 12.4 for the flowchart of Step 3.
REFERENCES Referenced Literature Laemmli, U. K. (1970). Nature, 227, 680–685.
Referenced Protocols in Methods Navigator Coomassie Blue Staining. Silver Staining of SDS-polyacrylamide Gel. Western Blotting using Chemiluminescent Substrates.
159
One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE) Julie L. Brunelle*,†, Rachel Green*,†,1
*Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA † Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, 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 Casting an SDS-PAGE Gel: Resolving Gel 5.1 Overview 5.2 Duration 5.3 Tip 5.4 Tip 5.5 Caution 6. Step 2 Casting an SDS-PAGE Gel: Stacking Gel 6.1 Overview 6.2 Duration 7. Step 3 Running an SDS-PAGE Gel 7.1 Overview 7.2 Duration 7.3 Tip References
152 152 153 153 154 154 154 154 154 154 155 155 155 155 155 156 157 157 157 159 159
Abstract This protocol describes a denaturing polyacrylamide gel system utilizing sodium dodecyl sulfate (SDS) to separate protein molecules based on size as first described by Laemmli (1970). SDS-PAGE can be used to monitor protein purifications, check the purity of samples, and to estimate molecular weights for unknown proteins.
Methods in Enzymology, Volume 541 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420119-4.00012-4
#
2014 Elsevier Inc. All rights reserved.
151
152
Julie L. Brunelle and Rachel Green
1. THEORY Polyacrylamide gel electrophoresis is useful for separating molecules by size and charge and there are many different systems depending on the sample and downstream applications. SDS-PAGE is a very useful tool to separate protein molecules by size. SDS is a detergent that denatures secondary and nondisulfide-linked tertiary structures and coats them with a negative charge that correlates with their length, allowing molecular weights to be estimated. Mobility through the gel can be affected by the state of the protein (e.g., phosphorylation and presence of multimeric molecules). The Laemmli SDS-PAGE system is a discontinuous gel with an upper stacking gel and lower resolving gel that have different pH values and polyacrylamide concentrations. The upper stacking gel has a lower percentage of polyacrylamide allowing proteins to move through quickly and ‘stack’ into a tight band before entering into the higher percentage polyacrylamide resolving gel for separation. The percentages of polyacrylamide can be optimized for the size range of molecules present in the sample. Gradient gels can be prepared allowing a greater range of separation in a single gel if both large and small proteins need to be resolved simultaneously. Small proteins will move through the resolving gel more quickly than large proteins. The protocol below is for mini gels, but can be scaled up for larger gel plates easily and directly. The method can be simplified greatly by using commercially available precast gels.
2. EQUIPMENT PAGE gel casting stand PAGE gel rig Glass plates Spacers Gel combs Power supply Pipet-aid Glass or disposable pipettes (5 ml and 10 ml) 10-ml syringe Needle (18–25 gauge) Micropipettors
153
One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE)
Micropipettor tips Gel loading tips 15-ml conical polypropylene tubes
3. MATERIALS Tris base Sodium dodecyl sulfate (SDS) Glycerol b-mercaptoethanol Bromophenol blue Glycine Hydrochloric acid (HCl) 40% acrylamide/bisacrylamide solution mix (19:1) Ammonium persulfate (APS) Tetramethylethylenediamine (TEMED) MilliQ H2O Protein molecular weight marker
3.1. Solutions & buffers Step 3 SDS-PAGE sample buffer Component
Final concentration
Stock
Amount
Tris–HCl, pH 6.8
120 mM
1M
0.6 ml
SDS
4%
10%
2 ml
Glycerol
20%
100%
1 ml
b-mercaptoethanol
750 mM
14.3 M
0.267 ml
Bromophenol blue
0.05%
Add water to 5 ml. Store in 0.5 ml aliquots at
0.0025 g 20 C
SDS-PAGE running buffer Component
Final concentration
Tris base
25 mM
3.025 g
Glycine
192 mM
14.4 g
SDS
0.1%
Add water to 1 l
Stock
10%
Amount
10 ml
154
Julie L. Brunelle and Rachel Green
4. PROTOCOL 4.1. Duration Preparation
5 min
Protocol
3–4 h
4.2. Preparation Clean the glass plates, spacers, and combs. Assemble the gel-casting sandwich and insert the comb to mark a line for the resolving gel height 1 cm below the bottom of the comb. See Fig. 12.1 for the flowchart of the complete protocol.
5. STEP 1 CASTING AN SDS-PAGE GEL: RESOLVING GEL 5.1. Overview Prepare and pour the lower 12% resolving gel.
5.2. Duration 1h 1.1 Place gel-casting sandwich into the casting stand. See Video 12.1, http://dx.doi.org/10.1016/B978-0-12-420119-4.00012-4. 1.2 Prepare the 12% resolving SDS gel solution:
Figure 12.1 Flowchart of the complete protocol, including preparation.
155
One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE)
Component
Final concentration
Stock
Amount
Acrylamide/bisacrylamide (19:1)
12%
40%
3 ml
SDS
0.1%
10%
0.1 ml
Tris–HCl, pH 8.8
375 mM
1.5 M
2.5 ml
Water
4.4 ml
1.3 Add 50 ml 10% APS and 5 ml TEMED; mix gently and quickly. See Video 12.2, http://dx.doi.org/10.1016/B978-0-12-420119-4.00012-4. 1.4 Pipette the resolving gel solution into the gel casting sandwich up to the line marked on the plate. 1.5 Overlay the gel with a layer of water-saturated butanol to smooth the top of the gel surface and aid in polymerization. 1.6 Allow the resolving gel to polymerize for 1 h. 1.7 Rinse the resolving gel with MilliQ water and remove any water droplets with a kimwipe. See Video 12.3, http://dx.doi.org/10.1016/ B978-0-12-420119-4.00012-4.
5.3. Tip APS and TEMED catalyze the polymerization of acrylamide. Once these are added, act quickly to pour the gel before it polymerizes.
5.4. Tip The percentage of acrylamide in the resolving gel can be adjusted to achieve the desired separation of proteins. 12% is used in this example and refers to the total concentration of acrylamide and bisacrylamide in the solution; 4–17% is the useful range for SDSPAGE.
5.5. Caution Acrylamide is a dangerous neurotoxin readily absorbed through the skin. Use gloves at all times. See Fig. 12.2 for the flowchart of Step 1.
6. STEP 2 CASTING AN SDS-PAGE GEL: STACKING GEL 6.1. Overview Prepare and pour the upper 5% stacking gel.
156
Julie L. Brunelle and Rachel Green
Figure 12.2 Flowchart of Step 1.
6.2. Duration 45 min 2.1 Prepare 5% stacking SDS gel solution: Component
Final concentration
Stock
Amount
Acrylamide/bisacrylamide (19:1)
5%
40%
0.623 ml
SDS
0.1%
10%
0.05 ml
Tris–HCl, pH 6.8
125 mM
1M
0.63 ml
Water
3.697 ml
2.2 Add 50 ml 10% APS and 10 ml TEMED; mix gently and quickly. See Video 12.4, http://dx.doi.org/10.1016/B978-0-12-420119-4.00012-4. 2.3 Pipette the stacking gel solution on top of the resolving gel in the gelcasting sandwich, filling to the top of the plate. 2.4 Insert the comb carefully to avoid trapping air bubbles; apply additional gel solution to fill in any low areas. 2.5 Allow the stacking gel to polymerize for 30–45 min.
One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE)
157
Figure 12.3 Flowchart of Step 2.
See Fig. 12.3 for the flowchart of Step 2.
7. STEP 3 RUNNING AN SDS-PAGE GEL 7.1. Overview Assemble gel rig and load samples to perform separation by applying electric current. See Video 12.5, http://dx.doi.org/10.1016/B978-0-12-420119-4. 00012-4.
7.2. Duration 1.5 h 3.1 Place the polymerized SDS-PAGE gel sandwich into the PAGE gel rig. 3.2 Add SDS gel running buffer to the upper and lower chambers. Check for bubbles along the bottom surface of the gel and remove by flushing with running buffer. 3.3 Remove the comb and rinse the wells with running buffer using a 10-ml syringe with a needle to flush away any unpolymerized acrylamide solution. 3.4 Dilute protein sample(s) at least 1:2 with SDS-PAGE sample buffer and heat at 95 C for 1–3 min.
158
3.5
Julie L. Brunelle and Rachel Green
Load molecular weight marker and prepared protein samples in the wells, using a micropipettor with gel loading tips. See Video 12.6, http://dx.doi.org/10.1016/B978-0-12-420119-4.00012-4. 3.6 Attach electrical leads to the gel rig and to the power supply matching the red and black. 3.7 Turn on the power supply to a constant 150–200 V for 35 min to 1 h. 3.8 When the bromophenol blue has run to the bottom of the gel, turn off the power supply and disconnect the leads. See Video 12.7, http://dx.doi.org/10.1016/B978-0-12-420119-4.00012-4. 3.9 Discard the running buffer and rinse the gel sandwich with water. 3.10 Carefully separate the glass plates to access the gel. 3.11 Proceed with downstream applications [e.g., staining (see Coomassie Blue Staining or Silver Staining of SDS-polyacrylamide Gel) or Western transfer (see Western Blotting using Chemiluminescent Substrates)].
Figure 12.4 Flowchart of Step 3.
One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE)
7.3. Tip 5–20 mg of protein can be detected by Coomassie blue staining. See Fig. 12.4 for the flowchart of Step 3.
REFERENCES Referenced Literature Laemmli, U. K. (1970). Nature, 227, 680–685.
Referenced Protocols in Methods Navigator Coomassie Blue Staining. Silver Staining of SDS-polyacrylamide Gel. Western Blotting using Chemiluminescent Substrates.
159
