CHEM 3501L Experiment:
Examining Protein Quaternary Structure Using Gel
Kennesaw State University, Kennesaw, GA 30144
±Quaternary Structure,² pp. 102-104.
Horton, Moran, Ochs, Rawn, and Scrimgeour.
of Biochemistry, 3
Prentice Hall, Upper Saddle River, NJ
Gel electrophoresis is a separation technique that separates molecules based on size and charge
In polyacrylamide gel electrophoresis (PAGE), protein samples are placed on a
highly cross-linked gel matrix and an electric current is applied.
polyacrylamide molecules act as molecular sieves, retarding the passage of large molecules more
than small molecules.
Gels can be made in the lab or bought from a supplier.
polyacrylamide gels come in a variety of concentrations, such as 7.5%, 12% and a gradient gel
The higher the percentage acrylamide, the smaller the pores will be.
Thus, the % gel
used is selected based on the protein size(s) in the sample of interest. You will be using the 4-
15% gradient gel because it allows for separation of a larger range of protein sizes (10 kDa ³250
In native PAGE techniques, protein migration is affected by size
determine a protein´s molecular weight, a method is needed in which charge does
protein to protein.
The technique that is typically used is SDS-PAGE.
When sodium dodecyl
sulfate (SDS), a negatively charged detergent, is added to the protein samples prior to placing on
the gel, the protein is denatured as the hydrophobic ends of SDS molecules associate with the
hydrophobic side chains of the amino acid residues in the protein.
The negatively charged end of
each SDS molecule points out toward the surface, as in a micelle arrangement, so each protein
molecule is surrounded by an overwhelming number of negative charges.
The migration of the
protein is now dependent on the molecular weight (size) of the protein.
Native PAGE techniques allow proteins to be separated in their native conformation.
the addition of SDS denatures the protein causing secondary, tertiary, and quaternary structure to
If a protein commonly containing two protein subunits held together by noncovalent
interactions is subjected to SDS-PAGE, the subunits should dissociate completely.
protein with a molecular weight of 100 kDa comprised of two identical chains of 50 kDa each
would appear as one single band in the loaded lane, with a molecular weight of approximately 50
Likewise, a protein with a molecular weight of 100 kDa made of two nonidentical chains
of molecular weights 30 kDa and 70 kDa would show up as two separate bands of 30 kDa and 70