1. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013
Practical 5: Protein Separation using Gel Electrophoresis: SDS-PAGE
Introduction
Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate
of migration depends on the strength of the field; on the nett charge, size and shape of the molecules and
also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As
an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the
properties of a single charged species, and as a separation technique.
SDS-PAGE stands for Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and is a
method useful in separation and identification of proteins according to their size. Since different proteins
with similar molecular weights may migrate differently due to their differences in secondary, tertiary or
quaternary structure, SDS which is an anionic detergent is used in SDS-PAGE to reduce proteins to their
primary (linearized) structure by breaking up the complex structure into their polypeptide subunits in the
process of denaturation of proteins. It then coats them with uniform negative charges by the process called
denaturation.
Generally, the procedures involve in SDS-PAGE are:
1) Making a gel and assembling the gel apparatus
2) Mixing protein samples with sample buffer containing SDS and heat the mixture at high temperature
3) Loading samples and running the electrophoresis
4) Fixing and staining the separated proteins.
Generally the sample is run in a support matrix which is polyacrylamide gel. The matrix inhibits convective
mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix
can be stained and used for scanning, autoradiography or storage. A porous gel may act as a sieve by
retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing
smaller molecules to migrate freely. Polyacrylamide is easier to handle when it is made at higher
concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore
size for retardation.
Materials, Reagents and Equipments
I) Mini-Protean Electrophoresis system Components
Spacer plate The Spacer Plate is the taller glass plate with gel spacers permanently
bonded. Spacer Plates are available in 0.5 mm, 0.75 mm, 1.0 mm, and
1.5 mm thicknesses, which are marked directly on each Spacer Plate.
Short plate The Short Plate is the shorter, flat glass plate that combines with the
Spacer Plate to form the gel cassette sandwich.
Casting frame The Casting Frame, when placed on the benchtop, evenly aligns and
secures the Spacer Plate and the Short Plate together to form the gel
cassette sandwich prior to casting.
Gel cassette assembly One Casting Frame, a Spacer Plate, and a Short Plate form one Gel
Cassette Assembly.
Casting stand The Casting Stand secures the Gel Cassette Assembly during gel
casting. It contains pressure levers that seal the Gel Cassette
Assembly against the casting gaskets.
Gel cassette sandwich A Spacer Plate and Short Plate with polymerized gel form a Gel
Cassette Sandwich after casting.
Combs A selection of molded combs is available.
Buffer dam The molded, one-piece buffer dam is used when running only one gel.
Electrode assembly The Electrode Assembly holds the Gel Cassette Sandwich. It houses
the sealing gasket, the upper and lower electrodes and the connecting
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2. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013
banana plugs. The anode (lower electrode) banana plug is identified
with a red marker and the cathode (upper electrode) banana plug with
a black marker.
Clamping frame The Clamping Frame holds the Electrode Assembly and Gel Cassette
Sandwich in place. Its pressure plates and closure cams seal the Gel
Cassette Sandwich against U-shaped gaskets on the Electrode
Assembly to form the inner buffer chamber.
Inner chamber The Electrode Assembly, two Gel Cassette Sandwiches or one gel
cassette sandwich and a buffer dam, and the Clamping Frame form
the Inner Chamber.
Mini tank and lid The Mini Tank and Lid combine to fully enclose the inner chamber
during electrophoresis. The lid cannot be removed without disrupting
the electrical circuit.
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3. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013
II) Sample loading buffer (Laemmli Loading dye) 3X stock
1M Tris-Cl, pH6.8 2.4ml
20% SDS 3.0ml
Glycerol 3.0ml
β-mercaptoethanol 1.6ml
Bromophenol blue 0.6g
Total volume is 10ml. Store at 4o
C.
III) 10X Running Buffer (also called Laemmli Buffer)
Tris Base (15g/l)
Glycine (72g/l)
SDS (1%)
Make to 1L with dH2O
IV) 1 M Tris-HCl, pH8.8
V) 1 M Tris-HCl, pH6.8
VI) 10% APS (ammonium persulphate)
VII) TEMED (N,N,N',N'-Tetramethylethylenediamine)
VIII) 0.25% (w/v) Coomassie Brilliant Blue R 250 in methanol-water-glacial acetic acid (5-5-1), filtered
immediately before use
VIIII) Samples (from Practical 4)
Methods
I) Gel cassette and casting stand assembly
Note: Ensure the casting stand, casting frames and glass plates are clean and dry before setting up the
casting stand assembly.
1. Place the Casting Frame upright with the pressure cams in the open position and facing on a flat
surface.
2. Select a Spacer plate of the desired gel thickness and place a Short Plate on top of it (see Figure
4a)
3. Orient the Spacer Plate so that the labeling is “up”. Slide the two glass plates into the Casting
Frame, keeping the Short Plate facing the front of the frame (side with pressure cams) (see Figure
4b).
Note: Ensure both plates are flush on a level surface and labeling on the Spacer Plate is oriented correctly.
Leaking may occur if the plates are misaligned or oriented incorrectly.
4. When the glass are up in place, engage the pressure cams to secure the glass cassette sandwich
in the Casting Frame (see Figure 4c). Check that both plates are flush at the bottom.
5. Engage the spring loaded lever and place the gel cassette assembly on the gray casting stand
gasket. Insure the horizontal ribs of the back of the Casting Frame are flush against the face of the
Casting Stand and the glass plates are perpendicular to the level surface. The lever pushes the
Spacer Place down against the gray rubber gasket (see Figure 4d).
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4. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013
II) Gel Casting
Discontinuous Polyacrylamide gels
1. Place a comb completely into the assembled gel cassette. Mark the glass plate 1cm below the
comb teeth. This is the level to which the resolving gel is poured. Remove the comb.
2. Prepare the resolving gel monomer solution by combining all reagents as follow:
Reagents Percentage of gel: 12.5%
37.5:1% w/v acrylamide:bisacrylamide 4.1 ml
1.0M Tris-Cl pH8.8 4.0 ml
20% SDS 50.7 ul
dH2O 1.73 ml
3. Add APS and TEMED to monomer solution and pour to the mark using a glass or disposable
plastics pipette.
Reagents Volume
10% APS 48.0 ul
TEMED 6.7 ul
4. Immediately overlay the monomer solution with water or butanol.
5. Allow the gel to polymerize for 45 minutes to 1 hour. Rinse the gel surface completely with
distilled water. Do not leave the alcohol overlay on the gel for more than 1 hour because it will
dehydrate the top of the gel.
Note: At this point the resolving gel can be stored at room temperature overnight. Add 5 ml of 1:4 dilution of
1.5 M Tris-HCl, pH8.8 buffer (for Laemmli System) to the resolving gel to keep it hydrated. If using another
buffer system, add 5ml of 1x resolving gel buffer to the resolving gel surface for storage.
6. Prepare the stacking gel monomer solution as follow:
Reagents Percentage of gel: 6.0%
37.5:1% w/v acrylamide:bisacrylamide 1.0 ml
1.0M Tris-Cl pH6.8 630 ul
20% SDS 25 ul
dH2O 3.0 ml
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5. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013
Note: Use a 4% of stacking gel if the resolving gel percentage is <10% and a 6% of stacking gel (easier to
handle) if the resolving gel percentage is >10%
7. Add APS and TEMED to the stacking gel monomer solution and pour the solution between the
glass plates. Continue to pour until the top of the short plate is reached.
Reagents Volume
10% APS 48.0 ul
TEMED 6.7 ul
8. Insert the comb between the spacers starting at the top of the Spacer Plate, making sure that
the tabs at the ends of each comb are guided between the spacers.
9. Allow the stacking gel to polymerize for 30-45 minutes.
10. Gently remove the comb and rinse the wells thoroughly with distilled water or running buffer.
III) Sample loading
1. Remove the Gel Cassette Assemblies from the Casting Stand. Rotate the cams of the Casting
Frames inward to release the Gel Cassette Sandwich (see Figure 5a).
2. Place a Gel Cassette Sandwich into the slots at the bottom of each side of the Electrode
Assembly. Be sure the Short Plate of the Gel Cassette Sandwich faces inward toward the
notches of the U-shaped gaskets (see Figure 5b).
3. Lift the Gel Cassette Sandwich into place against the green gaskets and slide into the Clamping
Frame (see Figure 5c).
4. Press down on the Inner Chamber and to insure a proper seal of the short plate against the
notch on the U-shaped gasket. (see Figure 5d). Short plate must align with notch n gasket.
Note: Gently pressing the top of the Electrode Assembly while closing the Clamping Frame cams forces the
top of the Short Plate on each Gel Cassette Sandwich to seat against the rubber gasket properly and
prevents leaking.
5. Lower the Inner Chamber Assembly into the Mini Tank. Fill the inner chamber with ~125 ml of
running buffer until the level reaches the halfway between the tops of the taller and shorter
glass plates of the gel Cassettes.
Note: Do not overfill the Inner Chamber Assembly. Excess buffer will cause the siphoning of buffer into the
lower chamber which can result in buffer loss and interruption of electrophoresis.
6. Add ~200 ml of running buffer to the Mini Tank (lower buffer chamber)
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6. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013
7. Mix your protein 4:1 with sample buffer and heat your sample by boiling for 5-10 minutes. Load
the samples into the wells with a pipette using gel loading tips.
IV) Electrophoresis
1. Apply power and begin electrophoresis at room temperature; 200 V constant until the
bromophenol blue is just off (approximately 50-60 minutes).
V) Visualization of Protein Separation
1. After electrophoresis, transfer the gel into a washing tray and rinse with dH2O twice.
2. Place the gel in at least 10 volumes of Coomasie Blue staining solution for 2-4 hours. Rock
gently to distribute the dye evenly over the gel.
3. At the conclusion of the staining, wash the gels with several changes of water.
4. Place the gels into a solution of 10% acetic acid for at least 1 hour.
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7. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013
5. If the background is still deeply stained at the end of the hour, move the gels to fresh 7% acetic
acid as often as necessary.
6. Photograph the gels or analyze the gels spectrophotometrically
Questions
1. What is the principle used in electrophoresis?
2. What is the function of SDS in this experiment?
3. In your opinion, which protein molecules are able to move faster in the gel: large macromolecules
or small molecules? Where do you think the positions of these molecules in the gel?
4. You are given a solution which contains a mixture of the six following proteins: Carbonic Albumin
from Bovine 66 kDa, Anhydrase from Bovine 29 kDa, β-Galactosidase from E. coli 116 kDa,
Albumin from egg 45 kDa, Phosphorylase B from Rabbit 97.4 kDa, Myosin from Rabbit muscle 200
kDa. Draw a schematic representation of the protein separation in a SDS-PAGE.
5. How would you prepare a 1.5 M of Tris-HCl, pH8.8 solution (Given the molecular weight of Tris-HCl
is 157.59)?
6. How would you prepare a 20% (w/v) SDS?
7. How would you prepare 1 litre of a running buffer which contains the following properties:
25mM Tris-HCl
200mM Glycine
0.1% (w/v) SDS
(Use the 1.5M Tris-HCl and 20% SDS solutions prepared in Question 5 and 6)
The molecular weight for Glycine is 75.07.
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