What is “electrophoresis”?
Electrophoresis is a technique whereby charged molecules are separated on the basis of their speed of migration in an applied electric field. Molecules that are cationic (that is, are positively charged) will migrate towards the negatively charged electrode (the cathode), while those molecules that are anionic (that is, are negatively charged) will migrate towards the positive electrode (the anode). The higher the charge, the faster will a molecule move towards the oppositely charged electrode.
If a mixture of charged molecules are placed in the center of a supporting medium (at the point S in the diagram on the right), and the electric field set up by means of an applied potential, V, then, after a while, separation of the molecules will take place.
Polynucleotides are generally anionic due to the presence of phosphate groups that are fully ionised at biological pH values. The charge on a protein will depend on the relative abundance of basic amino acids (lysine, arginine, histidine) and acidic amino acids (glutamic acid, aspartic acid), as well as the pH of the solution. At low pH-values, proteins will tend to be cationic, while at high pH values, proteins will tend to be anionic. There exists a pH value where the algebraic sum of positive and negative charges is zero, and the protein carries no net charge. This pH value is called the isoelectric point of the protein.
It can be shown that for a spherical molecule of mass M, the velocity v in a constant applied field, E, is directly proportional to that field and q, the charge on the molecule:
The above also implies that velocity is proportional to the mass to charge ratio, q/M.
The support matrix
Gel electrophoresis is commonly carried out on a slab of agarose. The dry substance is allowed to swell in hot buffer solution, and cast into a mould, which leaves small “wells” in the gel, into which the samples are applied. Other media that are used as support are: paper, cellulose acetate, starch, or polyacrylamide. For the latter, small slabs are prepared and run vertically in specially designed apparatus, to be described later.
The velocity, v, will also depend on the resistance to the movement of the molecules provided by the matrix through which the molecules are moving. Thus, the type of support that is used is very important. Agarose gels of various concentrations may be prepared by altering the ratio of dry agarose to the buffer. Typically, agarose gels are used in a concentration varying between 0.5% and 2%. Since molecular sieving takes place to varying extents, the more concentrated the gel, the slower the mobility of the molecules in the same buffer and applied potential difference.
Agarose gels are normally used to separate native proteins, that is, proteins that have retained higher orders of structure. One frequently refers to such gels as “native gels”. Separation on native gels takes place by both charge AND size. Polyacrylamide gels are normally used in conjunction with sodium docecyl sulphate.
What is “SDS-PAGE”?
SDS-PAGE stands for sodium docecyl sulphate-polyacrylamide gel electrophoresis. As its name implies, polyacrylamide is used a the support/matrix.
SDS and proteins
Sodium docecyl sulphate is a detergent that denatures proteins by
- unwinding helices,
- unfolding the proteins chains, and,
- separating protein chains.
The above processes are aided by the addition of mercaptoethanol (CH3CH2SH), which reduces intra- and intermolecular disulphide (-S-S-) bridges. As a consequence, the original protein is converted to a population of randomly coiled chains.
Further, SDS binds to the polypeptide chain, effectively coating the molecule with hydrophobic dodecyl residues, each of which carries a negative charge. The random coil conformation is converted to rod-like structures, as shown in the diagram above. SDS binds to proteins on a mass ratio basis: 1g of protein binds with 1.41 g SDS. Because the chains will effectively have the same mass to charge ratio, their electrophoretic mobility will be the about the same, and the successful separation of the proteins must depend on a sieving process in the gel.
Polyacrylamide gels are made by polymerising acrylamide monomer (CH2=CHCONH2) with ammonium persulphate [(NH4)2S2O8] in the presence of N,N’-methylene-bisacrylamide (“bis crosslinker”).
The resulting gel consists of minute “tunnels” of various diameters, which can selectively accomodate the passage of molecules, based on their sizes. There exists a linear relationship between the distance travelled in a given time under defined conditions and the logarithm of the molar mass of the proteins (diagram above, left). This is exploited in determining the molar mass of samples. The migration distance of unknown proteins is simply related to that of “markers” of known molecular masses. A schematic representation of this is shown in the diagram above on the right, where protein samples are electrophoresed in five “lanes” A-E, all migrating towards the positive pole of the electrophoretic cell:
Lane A consists of protein markers. Lane B consists of a mixture three proteins: x being the largest, and z the smallest, with y having a size intermediate between the two. Lane C, has pure protein x, lane D has pure y and lane E pure z.
For an improved separation in SDS-PAGE, a discontinuous, rather than continuous buffer system is preferred (A continuous system has only a single separating gel; and uses the same buffer in the tanks and the gel). In a discontinuous buffer system, a non-restrictive large pore gel, called a stacking gel, is layered on top of a separating gel called a resolving gel. Each gel is made with a different buffer, and the tank buffers are different from gel buffers.
Preparation of a discontinuous PAGE system requires casting two different layers of acrylamide between glass plates. The lower layer, the resolving, gel, is the gel that will perform the actual separation of proteins polypeptides by size. The upper layer is called the stacking gel, and it includes the sample wells. During electrophoresis, the proteins are compressed in the stacking gel into a very thin layer before they enter the resolving gel.
The stacking gel is
- more porous,
- is at a lower ionic strength, and
- at a lower pH
than the resolving gel. This causes the protein molecules to move faster in the stacking gel than in the resolving gel, because
- there is less physical resistance to the movement of protein molecules through the larger pores, and,
- the lower ionic strength in the stacking gel results in a higher electric field (V.cm-1).
The protein sample is normally applied in a TRIS/glycine buffer at pH 8.3. At that pH, there are three negatively charged species: the chloride ion, Cl -1, has a net charge of -1 at all pH values and is the smallest ion. It will therefore have the highest mobility. Glycine (pKa2 – 9.60), has a small net negative charge at pH 8.3, is larger than the chloride ion, and will therefore lag behind the chloride ions. Since the current carried by the electrolyte is the same through the gel system, the electric field is not the same in the chloride region as in the glycine region. The protein molecules have a charge that is intermediate between the chloride and the glycine ions, and concentrate in a very narrow band between the glycine and the chloride.
The stacking gel is at a pH of 6.8 At that pH, the proteins are appreciably anionic, while most of the glycine is present as the zwitterion which has no charge and hence will not migrate in the electric field. The proteins move rapidly through the stacking gel, and concentrate in a band between 1-100 μm thick, ahead of the glycine.
The resolving gel is at a pH of 8.8. At this pH, the glycine is almost completely anionic, and the proteins are now in a TRIS/glycine buffer (since the chloride ions have moved out). They move slowly, as the pores are much smaller. This results in a separation of the protein bands on the basis of the charge-to-mass ration of the proteins.
SDS-PAGE is very sensitive – as litle as 1×10-11 g of protein can be easily detected.
Electrophoresis and gel staining resources