GTB 204 Molecular Biology Protocols 2001

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 (β ME).

SDS disrupts the secondary, tertiary and quaternary structure of the protein to produce a linear polypeptide
chain coated with negatively charged SDS molecules. 1.4grams of SDS binds per gram of protein.
Mercaptoethanol assists the protein denaturation by reducing all disulfide bonds.

SDS-Polyacrylamide Gel Electrophoresis (PAGE)

Polyacrylamide gels are prepared by the free radical polymerization of acrylamide and the cross linking agent N
N’ methylene bis acrylamide

Acrylamide + N N’ methylene bis acrylamide

Chemical Ammonium persulfate (catalyst)

Polymerization +
TEMED (N,N N’ N’ tetramethylethylene diamine)

Polyacrylamide

Procedure:

1. Assembling the glass plate (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. Assemble the glass plate on a clean surface. Lay the longer glass plate down first, then place 2 spacers
of equal thickness along the rectangular plate. Next place the shorter glass plate on top of the spacers
so that the bottom ends of the spacers and glass plates are aligned (Figure 1).

2. Loosen the 4 screws on the clamp assembly and stand it up so that the screws are facing away from
you. Firmly grasp the glass plate sandwich with the longer plate facing away from you, and gently slide it
into the clamp assembly. Tighten the top 2 screws of the clamp assembly.

3. Place the clamp assembly into the alignment slot of the casting stand so that the clamp screws face
away from you. Loosen the top 2 screws to allow the plates and spacers to sit firmly against the casting
stand base. Gently tighten all the screws.

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4. Pull the completed sandwich from the alignment slot. Check that the plates and spacers are aligned. If
not, realign the sandwich as in steps 1-3. Before transferring the clamp assembly to the casting slot,
recheck the alignment of the spacers. Do this by inverting the gel sandwich and looking at the surface of
the 2 glass plates and the spacer. Make sure that they are aligned.

5. Transfer the clamp assembly to one of the casting slots in the casting stand. If 2 gels are to be prepared,
place the clamp assembly on the other side of the alignment slot.

6. Press the acrylic pressure plate bottom, so that the glass plates rest on the rubber gasket. Snap the
acrylic plate underneath the overhang of the casting slot. Do not push the glass plates or spacers
because this could break the glass plate.

Figure 1

2. Casting the gels (Demonstration)

Prepare 10% 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.

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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.

10. Remove the comb.

11. Gel is placed in the buffer chamber and running gel buffer is added into the chamber

3. Preparation of samples (By students)

From the recombinant clone VC-25, the recombinant protein is produced as follows

The clone was grown for 4hours and induced using IPTG for next four hours. The culture was pelleted and
resuspended in PBS

Each group is provided with the following

Procedure Tube 1 Tube 2 Tube 3 Tube 4

Mol. Wt Vector
Uninduced Induced clone
Content Marker protein
clone protein protein

Sample buffer
(Glycerol, SDS,
- 20µl 20µl 20µl
Mercaptoethanol,
Bromophenol blue dye)
Do not boil
Boil for 5 min Boil for 5 min Boil for 5 min Boil for 5 min

Not
Brief spin 5 min 5 min 5 min
required
Loading sample into the
10µl 20µl 20µl 20µl
well

4. Loading the samples (By students)

1. Rinse the syringe to be used for loading samples a few times with distilled water. Demonstrators will
load the first well with LMW (7 µl of LMW). Insert the syringe to about 1-2 mm from the well bottom
before delivery. Rinse the syringe a few times with distilled water after loading.

2. Load the second and other well with 20 µl of VC-25 protein as described above. Do not pipette the
pellet at the bottom of the microfuge tube. Rinse the syringe a few times with distilled water after loading.

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5. 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.

6. Removing and staining the gel (By tutor)

1. Remove the gel from the buffer chamber

2. Loosen all four screws of the clamp assembly and remove the glass plate sandwich from it.

3. Push one of the spacers out to the side of the plates without removing it.

4. Gently twist the spacer so that the upper glass plate pulls away from the gel.

5. Cut the gel on one side (to orientate the gel).

6. Remove the gel by gently grasping two corners of the gel and place it in the container containing the
Coomassie blue stain. Make sure that the gel is fully submerged in the staining solution.

7. Stain the gel for 1 hour, agitate it slowly on a shaker.

8. Destain the gel in a destaining solution a few times until protein bands are visualised.

9. Approximately determine the molecular weight of the visualised protein bands by comparing them
with the molecular weight markers.

Appendix 1

Preparation of a 10% Resolving/separating gel.

30% acrylamide + 0.8% Bis-acrylamide 1.25 ml

4X Tris-HCl/SDS, pH 8.8 0.94 ml
Distilled water 1.56 ml
10% ammonium persulfate (APS) 25 µl
TEMED 10 µl

Preparation of a 4.5% stacking gel

30% Acrylamide + 0.8% Bis-acrylamide 0.65 ml

4X Tris-HCl/SDS, pH 6.8 1.25 ml
Distilled water 3.05 ml
Ammonium persulfate 25 µl
TEMED 5 µl