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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.
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.
Figure 1

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

Reference GTB 204 Molecular Biology Protocols 2001


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