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: ragreen@jhmi.edu

Contents
1. Theory 152
2. Equipment 152
3. Materials 153
3.1 Solutions & buffers 153
4. Protocol 154
4.1 Duration 154
4.2 Preparation 154
5. Step 1 Casting an SDS-PAGE Gel: Resolving Gel 154
5.1 Overview 154
5.2 Duration 154
5.3 Tip 155
5.4 Tip 155
5.5 Caution 155
6. Step 2 Casting an SDS-PAGE Gel: Stacking Gel 155
6.1 Overview 155
6.2 Duration 156
7. Step 3 Running an SDS-PAGE Gel 157
7.1 Overview 157
7.2 Duration 157
7.3 Tip 159
References 159

Abstract
This protocol describes a denaturing polyacrylamide gel system utilizing sodium dode-
cyl 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 # 2014 Elsevier Inc. 151

ISSN 0076-6879 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-420119-4.00012-4
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 sep-
arate 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 pro-
tein (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 polyacryl-
amide 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 pro-
tocol 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
One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE) 153

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% 0.0025 g
Add water to 5 ml. Store in 0.5 ml aliquots at 20  C

SDS-PAGE running buffer

Component Final concentration Stock Amount
Tris base 25 mM 3.025 g
Glycine 192 mM 14.4 g
SDS 0.1% 10% 10 ml
Add water to 1 l
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 sand-
wich 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.

One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE) 155

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 drop-
lets 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 SDS-
PAGE.

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 gel-
casting 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 acryl-
amide 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 Julie L. Brunelle and Rachel Green

3.5 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) 159

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.