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Revised 6.20.

05

SDS-PAGE: Laemmli System


Solutions
1. Upper Gel Buffer (4X): 0.4% (w/v) SDS in 0.5 M Tris-HCl, pH 6.8. Store at 4C.
2. Lower Gel Buffer (4X): 0.4% (w/v) SDS in 1.5 M Tris-HCl, pH 8.8. Store at 4C.
3. Acrylamide/Bis-Acrylamide (A/BA) Solution: We use the BioRad version, which is 30%
(w/v) total, in a 37.5:1 ratio (2.6% [w/v] crosslinker), Catalog #161-0158.
4. Fresh 10% (w/v) ammonium persulfate (APS) in ddH2O: we use the BioRad persulfate
(Catalog #161-0700), which is stored at room temperature in a degassed chamber. Please,
pump the air out when closing the chamber. Keep in ice until use.
5. TEMED: we use the BioRad TEMED solution (Catalog #161-0801); the manufacturer advice
to store at room temperature, although it sediments under these conditions (without change in its
efficiency as a catalyst for polymerization). Some people have found that storing it at 4C
decreases sedimentation.
6. Fresh 1 M dithiotreitol (DTT) in ddH2O (for reducing conditions) or 1 M N-ethylmaleimide
(NEM) in absolute ethanol (for non-reducing conditions). Use only Sigma DTT or NEM; it is
very important to make them fresh since they oxidize easily. Both reagents are stored at 20 C.
Keep in ice until use.
7. Sample Denaturing Buffer (5X): 10% (w/v) SDS, 50% (v/v) glycerol, and 0.005% (w/v)
bromophenol blue in 310 mM Tris-HCl, pH 6.8. Store at room temperature.
8. Laemmli Gel Running Buffer (5X): 0.5% (w/v) SDS, 126 mM Tris, and 0.96 M glycine. Store
at room temperature.
Procedure (for two gels)
1. Set up casting stands for BioRad Mini-Protean II or III. Generally, we make 1.5 mm thick
gels, since the Ryanodine receptor subunit (and the oxidized dimers) enters better to the gel
when they are thick. But there are also 0.75 mm thick spacers (and spacer glass plates) in the

lab. Right before casting the gels, the glass plates should be washed with soap by hand (do
not use brushes, since they scratch the glass) and rinsed with 20% ethanol, or using the Gel
Plate Solutions I and II (from Geno Technology, Inc., Catalog #786-140 for spraying bottles
and #786-140RF for refills).
2. The 5X sample denaturing buffer is very dense, so it is good at this point to heat it up a little;
this can be accomplished either in a heated block between 50-60C or in a microwave for a
few seconds (be careful not to flood!).
3. The following recipes are enough for 2 x 1.5 mm thick gels:
3a) Resolving (lower) gel: In a 50-mL Falcon tube, mix the following reagents:
DESIRED FINAL %

5%

7.5%

10%

12.5%

10

12

10

15%
30% A/BA solution (mL)

4
12

4X Lower Gel Buffer (mL)

6
6

ddH2O (mL)

14
6

Vortex and add 120 L 10% APS; vortex and add 12 L TEMED; vortex and pour monomer
(8 mL per gel). Carefully, pour some ddH2O on top of the monomer to insulate from air (O2
inhibits polymerization) and to get bubbles out, as well as making the top of the gel smooth
and straight. Let polymerize for at least 30 min at room temperature. Once polymerization
has taken place (a very sharp interface has formed between the gel and the water layer),
discard water and dry plates carefully with paper towels.
3b) Concentrating (upper) gel: In a 15-mL Falcon tube, mix 1.6 mL 30% A/BA solution, 3
mL 4X Upper Gel Buffer and 7.4 mL ddH2O. Vortex and add 90 L 10% APS; vortex and
add 20 L TEMED; vortex and pour enough monomer to fill out the rest of the plates.

Introduce the combs immediately, displacing the excess of monomer with the introduction of
the comb. This must be accomplished quickly, since this gel polymerizes very fast.
4. Denaturation of samples: Add fresh DTT (for reducing conditions) or NEM (for nonreducing conditions) to the 5X sample denaturing buffer to reach final 125 mM DTT or 25
mM NEM. Dilute the sample denaturing buffer with sample and ddH2O to reach 1X sample
denaturing buffer and the desired protein quantity in less than 35 L. For SR vesicle
preparations, usually 10 to 20 g protein is enough to see a profile without overloading the
gel. For other samples, different protein quantities might be needed. Let samples sit for 1
hour at room temperature or 30 min at 50 to 60C.
5. Loading: Dilute 200 mL of 5X Running Buffer with ddH2O (1 L final). Take out the combs
from the plates carefully not to disturb the wells. Pour some diluted Running Buffer on the
wells. Load samples (for gels 1.5 mm thick, up to 35 L can be loaded per well without
sample flooding to neighbor wells). Dont forget to run one lane of MW standards.
6. Running the electrophoresis: Set up the gels into the running chamber and cover the entire
chamber with diluted Running Buffer. Cover with the lid, carefully not to confuse the
electrode colors. Set up the run for 80 V constant for the first 30 min; then, the run can be
either left at 80 V constant or increase voltage to 120 V constant, or run with 30-35 mA
(constant current) per gel. Run until the front reach the end of the gel (about 1.5-3 hours,
depending on the settings). Usually, best results are obtained when the electrophoresis is run
in the cold room, or the camber is in an ice bucket.