The McGill Physiology Virtual Lab

Biochemical/molecular techniques

Background concepts:

Gel electrophoresis is a technique which separates macromolecules - nucleic acids or proteins- on the basis of size, electric charge and other physical properties.
 SDS-Polyacrylamide gel electrophoresis (PAGE) further resolves complex mixtures of proteins by using the ability of a detergent SDS (sodium dodecyl sulfate) to denature and reduce polypeptides to flexible rods with uniform negative charge per unit length. Since molecular weight is now a linear function of peptide length, the proteins separate by molecular weight through the pores of the gel. The smaller the molecule, the faster it separates.
The purpose is to separate or isolate proteins from a crude sample, estimate the purity and the molecular weight of a protein in kilodaltons (kDa).

The technique
Electrophoresis is the migration of charged molecules in solution inside a support matrix (a colloid in a solid form) subjected to an electrical field (between a negative and a positive electrode). Here, polyacrylamide gel will be used as a support matrix: it is used to separate most proteins and small oligonucleotides requiring a small gel pore size. Polyacrylamide gel is polymerized from a solution of acrylamide monomers into cross-linked chains, forming a semi solid matrix.
 The rate at which the proteins move in the gel depends on the electric current, the pH of the medium, the pore size of the gel, the friction which in turn depends on the shape and size of the protein (smaller molecules will move faster through the pores of the gel).


Considerations over the separation of proteins:
The net charge of a protein depends on the pH of its environment. In a solution with a pH above its isoelectric point, a protein has a net negative charge and will migrate towards the positive electrode. Below the isoelectric point, the protein is positively charged and therefore migrates towards the negative electrode. It is important to note that the net charge carried by a protein is also independent of its size: the charge carried per unit mass of molecule is different from protein to protein.
At a given pH and under non-denaturing conditions, the electrophoretic migration of proteins is determined by size and charge of the molecules.

  • If the matrix is buffered to weakly basic, most proteins loaded on the gel will be anionic and migrate to the positive electrode when an electric current is applied.
  • If one chooses to limit the migration of proteins to only one factor, namely protein size or mass, then further treatment of the proteins by an anionic detergent (sodium dodecyl sulfate or SDS) must be considered.

Migration of proteins under denaturing conditions:
SDS is an ionic detergent with a long hydrophobic tail which binds to the hydrophobic side chains of aminoacids at a constant ratio of 1.4 g of SDS per gram of polypeptide. This ratio ensures that all SDS treated proteins have a similar mass to charge ratio; so, net protein charge is no longer a factor affecting its migration. SDS also denatures proteins, dissociates multimeric proteins into subunits and causes extended conformations ("flexible rods") in the polypeptides. As a result,  shape of the protein is no longer a factor in its migration, but its molecular weight is.

The picture above shows gels that  have been mounted in one electrophoresis apparatus and which will receive samples  for protein migration and separation. Special long tips are used to facilitate loading.

The comb is gently removed to allow the running buffer to enter the wells. The samples are loaded slowly, into separate wells, with the pipette tip close to the bottom of the well without touching the gel.


The proteins, stained with a fluorescent dye, are loaded into the wells of the gel and electrophoresed. Bands appear on the gel, showing the migration distances of different sized proteins. Migration distances are characteristic to specific proteins and are used to assess the purity of protein. If the protein under study is unidentified, it can be compared to the migration distances of a known protein (standard) for an estimation of its molecular weight. The gel concentration must be chosen so that the standards give out a linear curve in the region of the unknown.

For an interesting electrophoresis simulation from the Rochester Institute of Technology, click here

to continue with the next section: spectrophotometry, click here