SDS-PAGE Ms. Nadia Amara

Electrophoresis is a process in which molecules are exposed to an electric field and separated on the basis of their differential mobilities in that field. SDS-PAGE, (sodium dodecyl sulfate polyacrylamide gel electrophoresis): is a technique widely used in biochemistry, forensics, genetics and molecular biology, to separate proteins according to their size (length of polypeptide chain or molecular weight). The separation of proteins usually involves the use of polyacrylamide as the matrix. Polyacrylamide is formed by polymerization of acrylamide monomers in the presence of N,N´-methylene bis-acrylamide.

The polymerization reaction shown in the diagram below, catalyzed by free radicals. The reaction is initiated by (Tetramethylethylenediamine) (TEMED), which induces free radical formation from unstable compound ammonium per sulphate (APS).

The free radicals transfer electrons to the acrylamide/bis acrylamide monomers, radicalizing them and causing them to react with each other to form the polyacrylamide chain. In the absence of bis-acrylamide, the acrylamide would polymerize into long strands (linear polymer ), not a porous gel. But as the diagram shows, bis-acrylamide contains two double bonds, which allow the compound to act as across-linker between acrylamide chains and this is what gives rise to the formation of the porous gel matrix.

Varying the amount of acrylamide monomer and bis-acrylamide cross-linker present controls the formation of the matrix. The use of larger amounts of these components results in a denser matrix. Denser matrices are used for separating smaller proteins; larger proteins may find the pores in a dense matrix too small to enter, and may therefore not enter the gel at all.

Real proteins have different charged side-chains Real proteins have different charged side-chains. As a result, real proteins do not have constant charge-to-mass ratios . Real proteins also have varying three-dimensional shapes. In order to measure molecular weight, it is necessary to induce the formation of a similar shape and charge-to-mass ratio. Boiling the protein in the presence of the detergent sodium dodecyl sulfate (SDS) and the reducing agent b-mercaptoethanol (which reduces disulfide bonds) results in disruption of the three dimensional structure of the protein.

SDS (the detergent soap) breaks up hydrophobic areas and coats proteins with negative charges thus overwhelms any intrinsic charge present in the protein. The detergent binds to hydrophobic regions in a constant ratio of about 1.4 g of SDS per gram of protein. (approximately one molecule of SDS for every two amino acid residues).

Note that treatment with SDS and b-mercaptoethanol will result in the formation of denatured protein monomers; it is these protein monomers that are separated on the SDS PAGE. The molecular weight standards can be used to calibrate the migration of proteins of differing sizes on the gel. Note that proteins within an SDS polyacrylamide gel are denatured; the molecular weight determined will be that of the individual monomers of multimeric proteins.

As mentioned above, when running an SDS PAGE, you must denature your proteins and coat them with SDS. This is usually done by boiling the protein sample in SDS PAGE sample buffer. The sample buffer has three roles: The sample buffer provides the SDS necessary for the uniform charge-to-mass ratio. The sample buffer also provides a tracking dye (usually bromophenol blue, a blue dye that runs at a low apparent molecular weight). The tracking dye makes it easier to see the sample during loading, and lets you know when the lowest molecular weight proteins are getting near the end of the gel.

Finally, the sample buffer contains glycerol to make the protein sample denser than the electrophoresis tank buffer, so that the protein sample will sink to the bottom of the well when you load it. The type of SDS polyacrylamide gel you will use consists of two layers: The top layer is the stacking gel and the bottom layer is the resolving gel (separating gel ) , based on a method invented by Laemmli in 1970. The purpose of the two sections differs : the stacking gel concentrates all of the protein in a narrow region, while the resolving gel performs the actual separation of the proteins by molecular weight. The stacking gel also contains the wells into which the samples are loaded.

The stacking gel is prepared at a lower pH (6 The stacking gel is prepared at a lower pH (6.8) and lower acrylamide percentage . What if not using a stacking gel? In the absence of a stacking gel, your sample would sit on top of the running gel, as a band of up to 1cm deep rather than being lined up together and hitting the running gel together, the proteins in the sample would all enter the running gel at different times, resulting in very smeared bands . So the stacking gel, ensures that all of the proteins arrive at the running gel at the same time so proteins of the same molecular weight will migrate as tight bands.

The resolving gel ( Separation) is at pH 8 The resolving gel ( Separation) is at pH 8.8 and has the desired acrylamide concentration for separation of proteins in the appropriate size range . Once the proteins are in the running gel, they are separated because higher molecular weight proteins move more slowly through the porous acrylamide gel than lower molecular weight proteins.

The role of Glycine : Glycine can exist in three different charge states, positive, neutral or negative depending on the pH. Control of the charge state of the glycine by the different buffers is the key to the whole stacking gel thing.

The Way stacking gel works.. When the power is turned on: Negatively-charged glycine ions in the pH 8.3 electrode buffer are forced to enter the stacking gel, where the pH is 6.8. In this environment glycine switches predominantly to the zwitterionic (neutrally charged) state. This loss of charge causes them to move very slowly in the electric field. The Cl- ions (from Tris-HCl) on the other hand, move much more quickly in the electric field and they form an ion front that migrates ahead of the glycine.

3. The SDS-coated protein molecules and the dye, which have charge-to-mass ratios greater than that of the glycine but less than that of Cl, must migrate behind the Cl and ahead of the glycine. This has the effect of concentrating the proteins in a thin band sandwiched between the Cl ions and the glycine molecules. All of the proteins in the gel sample have an electrophoretic mobility that is intermediate between the extreme of the mobility of the glycine and Cl- . In addition, because the acrylamide concentration of the stacking gel is very low most proteins are not retarded and move freely through the gel matrix. When the sample reaches the end of the stacking gel it should appear as a thin blue band.

4. This procession carries on until it hits the running gel, where the pH switches to 8.8. At this pH the glycine molecules are mostly negatively charged and can migrate much faster than the proteins. So the glycine front accelerates past the proteins, leaving them behind. Since the running gel has an increased acrylamide concentration, which slows the movement of the proteins according to their size, the separation begins…

The end result of SDS-PAGE has two important features: all proteins contain only primary structure all proteins have a large negative charge which means they will all migrate towards the positive pole when placed in an electric field.