SDS-PAGE

Principle
Electrophoresis is the study of the movement of charged molecules in an electric field. The
generally used support medium is cellulose or thin gels made up of either polyacrylamide or
agarose. Cellulose is used as support medium for low molecular weight biochemicals such as
amino acid and carbohydrates whereas agarose and polyacrylamide gels are widely used for
larger molecules like proteins. The general electrophoresis techniques cannot be used to
measure the molecular weight of the biological molecules because the mobility of a substance
in the gel is influenced by both charge and size. In order to overcome this, if the biological
samples are treated so that they have a uniform charge, electrophoretic mobility then depends
primarily on size. The molecular weight of protein maybe estimated if they are subjected to
electrophoresis in the presence of a detergent sodium dodecyl sulfate (SDS) and a reducing
agent mercaptoethanol (ME). SDS disrupts the secondary, tertiary and quaternary structure
of the protein to produce a linear polypeptide chain coated with negatively charged SDS
molecules. All proteins are now negatively charged with similar charge density and thus can
be separated on the basis of their size only. 1.4grams of SDS binds per gram of protein.
Mercaptoethanol assists the protein denaturation by reducing all disulfide bonds.
SDS-PAGE is used mainly for the following purpose:
a. Estimation of protein size.
b. Determination of protein subunits or aggregation structures.
c. Estimation of protein purity.
d. Protein quantitation.
e. Monitoring protein integrity.
f. Comparison of the polypeptide composition of different samples.
g. Analysis of the number and size of polypeptide subunits.
h. Post-electrophoresis applications, such as Western blotting.

Procedure:
1.

Gel Cassette Sandwich Preparation (Demonstration)

Note:
1. Gloves should be worn at all times while performing SDS-PAGE.
2. To insure proper alignment and casting, the glass plates, spacers, combs and casting stand
gaskets must be clean and dry. The glass plates should be cleaned with 70% ethanol.

1. Glass 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. During regular use, a powder residue may build up behind the pressure
cams of the casting frame at the pivot point. This powder should be removed before each use.

a. Place the Casting Frame upright with the pressure cams in the open position and facing
forward on a flat surface.
b. Select a Spacer Plate of the desired gel thickness and place a Short Plate on top of it

c. 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)
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.

d. When the glass plates are in place, engage the pressure cams to secure the glass cassette
sandwich in the Casting Frame. Check that both plates are flush at the bottom. Engage the
spring loaded lever and place the gel cassette assembly on the gray casting stand gasket.
Insure the horizontal ribs on 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.
e. Repeat steps ad for a second gel.

Figure 1. Mini-PROTEAN 3 system components.

2.

Casting the gels (Demonstration)

Prepare 12.6 % resolving/separating gel and 4.5% stacking gel. Please refer to appendix 1 for
the recipe.
1. Prepare the separating gel monomer solution by combining all reagents except
ammonium persulfate (APS) and TEMED. Deaerate and mix the solution after adding each
reagent by swirling the container gently.
2. Place a comb completely into the assembled gel sandwich. With a marker pen, place a
mark on the glass plate 1 cm below the teeth of the comb. This will be the level to which the
separating gel is poured. Remove the comb.

3. Add APS and TEMED to the monomer solution and mix well by swirling gently. Pipette the
solution to the mark.
4. Immediately overlay the monomer solution with 1 ml. of water. Use a steady, even rate of
delivery to prevent mixing with the gel.
5. Allow the gel to polymerize for 45 minutes to 1 hour. Pour the water overlaying the gel and
drain the excess water with strips of filter paper.
6. Prepare the stacking gel monomer solution. Combine all reagents except APS and
TEMED. Deaerate and mix the solution by swirling gently.
7. Place a comb in the gel sandwich.
8. Add APS and TEMED to the solution and pipette the solution down one of the spacer until
the sandwich is filled completely.
9. Allow the gel to polymerize for 15 minutes.

3. Preparation of samples (By tutor)

The clone was grown for 4hours and induced using IPTG for next four hours. The culture was
pelleted and resuspended in samples buffer. Please refer to appendix 1 for the recipe.

4. Mini-PROTEAN 3 Electrophoresis Module Assembly (Demonstration)

1. Remove the Gel Cassette Assemblies from the Casting Stand. Rotate the cams of the
Casting Frames inward to release the Gel Cassette Sandwich
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
3. Lift the Gel Cassette Sandwich into place against the green gaskets and slide into the
Clamping Frame
4. Press down on the Electrode Assembly while closing the two cam levers of the Clamping
Frame to form the Inner Chamber and to insure a proper seal of the short plate against the
notch on the U-shaped gasket. Short plate must align with notch in 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 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).

5. Loading the samples (By students)

1. Rinse the well a few times with running gel buffer using the syringe. Load the samples into
the wells with a pipette using gel loading tips. Demonstrators will load the first well with LMW
(7 l of LMW). Insert the tip (10 l) to about 1-2 mm from the well bottom before delivery.

2. Load the second and other well with 20 l of protein sample as described above. Do not
pipette the pellet at the bottom of the microfuge tube.
Note: Load samples slowly to allow them to settle evenly on the bottom of the well. Be careful not to
puncture the bottom of the well with the syringe needle or pipette tip.

6. Running the gel

1. Check that the buffer in the upper buffer chamber are full because leakage of the buffer
may occur.
2. Place the lid on top of the lower buffer chamber. Make sure that the connection is correct,
ie. black to black and red to red.
3. Attach the electrical leads to a suitable power pack with the proper polarity (black to black
and red to red). Run the gel at a constant current of 30 mA.
4. Stop the electrophoresis when the tracker dye is ~ 1 cm above the end of the glass plates.

7. Removing and staining the gel (By tutor)

Gel Removal
1. After electrophoresis is complete, turn off the power supply and disconnect the electrical
leads.
2. Remove the tank lid and carefully lift out the Inner Chamber Assembly. Pour off and discard
the running buffer.
Note: Always pour off the buffer before opening the cams to avoid spilling the buffer.
3. Open the cams of the Clamping Frame. Pull the Electrode Assembly out of the Clamping
Frame and remove the Gel Cassette Sandwiches.
4. Remove the gels from the Gel Cassette Sandwich by gently separating the two plates of
the gel cassette. The green, wedge shaped, plastic Gel Releaser may be used to help pry the
glass plates apart.
Note: To remove the gel from a Ready Gel Cassette, first slice the tape along the sides of the
Ready Gel Cassette where the inner glass plate meets the outer plastic plate.
5. Run the sharp edge of the Gel Releaser or a razor blade along each spacer to separate the
gel from the spacer. Remove the gel by floating it off the glass plate by inverting the gel and
plate under fixative or transfer solution, agitating gently until the gel separates from the plate.
6. Rinse the Mini-PROTEAN 3 cell electrode assembly, Clamping Frame and Mini Tank with
distilled, deionized water after use.
Gel staining
4. Place the removed gel in the container containing the Coomassie blue stain. Make sure
that the gel is fully submerged in the staining solution.
5. Stain the gel for 1 hour, agitate it slowly on a shaker.
6. Destain the gel in a destaining solution a few times until protein bands are visualized.
7. Approximately determine the molecular weight of the visualized protein bands by comparing
them with the molecular weight markers.

Appendix
Preparation of a 12.6 % Resolving/separating gel. 4.5 ml/minigel
30% acrylamide + 0.8% Bis-acrylamide 1.89 ml
1.5 M Tris-HCl, pH 8.8, 1.125 ml
Distilled water 0.55 ml
10% ammonium persulfate (APS) 25 ml
10% SDS 45 ml
TEMED 3 ml
50% Glycerol
Preparation of a 4.5% stacking gel, 0.8 ml
30% Acrylamide + 0.8% Bis-acrylamide 0.14 ml
0.5MTris-HCl, pH 6.8 0.2 ml
Distilled water 0.45 ml
10% SDS 4 ml
Ammonium persulfate 20 ml
TEMED 1 ml
Use the following guide to determine % acrylamide in resolving gel:
5% for SDS-denatured proteins 60 to 200 kDa
7.5% for proteins 50 to 120 kDa
10% for proteins 16 to 70 kDa
12.5% for proteins 15 to 60 kDa
15% for proteins 12 to 45 kDa
The % acrylamide in the stacking gel is constant.
Molecular Weight Markers
PageRuler Prestained Protein Ladder (Fermentas cat# SM0431) and find this to be a good
broad-range marker for proteins 14 to 116 kDa. Load 5 l per lane for 10-well comb.
Coomassie Stain (1 L)
0.1% coomassie R-250
40% ethanol
10% acetic acid
To 480 ml water add 420 ml 95% ethanol and 100 ml acetic acid while stirring. Add 1.0 g
coomassie R-250. Transfer to storage bottle that has been rinsed to remove any precipitate.
6 Sample Buffer
Combine the following in a 15 ml conical tube and gently agitate on rotator until components
are dissolved. For 10 ml:
7 ml 0.6 M Tris-Cl pH 6.8
3 ml glycerol
1 g SDS
1.2 mg bromophenol blue
10Running Buffer
60.6 g Tris base
288 g Glycine

Dissolve in 1600 ml water and bring the volume to 2 litres. Transfer to a 2 L bottle using a
funnel. Carefully add 20 g SDS while stirring. Adding the SDS before transfer to the bottle will
result in bubbles, making transfer impossible. Dilute 50 ml of this stock with 450 ml water for
each electrophoresis run and mix thoroughly.