Due to the attributes and interactions of amino acid residues, proteins have unique characteristics that can be isolated and analyzed by separation techniques.
The pH of an aqueous solution can affect the solubility of the solute. By changing the pH of the solution, you can change the charge state of the solute. If the pH of the solution is such that a particular molecule carries no net electric charge, the solute often has minimal solubility and precipitates out of the solution. The pH at which the net charge is neutral is called the isoelectric point (pI).
Amino acids all contain the same backbone, which has both an acidic (-COOH) and a basic (-NH2) group. Each amino acid also has a functional group attached to the backbone. These functional groups can be positive, negative, neutral, or polar in nature. The backbone and functional groups give a protein its overall charge. At a pH below the protein’s pI, a protein will carry a net positive charge; above its pI, it will carry a net negative charge. Proteins can therefore be separated according to their isoelectric point.
In a method called isoelectric focusing, proteins are run through a gel that has a pH gradient. The gels are set in a buffer in a container with a negatively charged electrode (cathode) on one end and a positively charged electrode (anode) on the other. When the proteins are added to the solution and current is applied, they migrate toward the electrode with the opposite charge.
Gel electrophoresis is a technique used to separate molecules based on size and charge, and can be used to separate proteins as whole molecules or fragments. The proteins/peptides are loaded into a slot near the negative electrode of a the porous gel matrix. Proteins are most commonly run in polyacrylamide gel electrophoresis (PAGE) gels. Because proteins are generally negatively charged, they are pulled toward the positive electrode at the opposite end of the gel. Smaller molecules move through the pores in the gel faster than larger molecules, and this difference in the rate of migration separates the fragments based on size. There are molecular weight standard samples that can be run alongside the molecules to provide a size comparison.
Proteins in their natural, tertiary-structured state can be run on native-PAGE gels – this separates proteins by both size and conformation. Larger, bulkier proteins will run toward the positive cathode more slowly. Proteins can also be chemically denatured before running them on the gel – in this case, proteins are separated exclusively by size. The chemical SDS denatures proteins, and so denaturing SDS-PAGE gels are used for these experiments. Proteins can be further denatured by the addition of a reducing agent that breaks down the disulfide bonds between cysteine residues (these are not fully broken by SDS alone).
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• If the pH of the solution is such that a particular molecule carries no net electric charge, the solute often has minimal solubility and precipitates out of the solution.
• The pH at which the net charge of the solute is neutral is called the isoelectric point.
• At a pH below a molecule’s pI, that molecule will carry a net positive charge; at a pH above its pI, the molecule will carry a net negative charge.
• Isoelectric focusing can be used to separate different compounds in a mixture, particularly proteins.
• Gel electrophoresis can be done using native-PAGE or SDS-PAGE (with or without a reducing agent), depending on whether you are interested in the native conformation or the denatured state of the protein.
isoelectric point: pH at which the net charge of a molecule is neutral
isoelectric focusing: a technique for separating proteins based on their isoelectric point (pI)
gel electrophoresis: separation of molecules based on size and charge
native-PAGE: a technique for separating proteins based on size and conformation
SDS-PAGE: a technique for separating proteins based on size only, because proteins are denatured
reducing: refers to the chemical reduction of the disulfide bonds between cysteine residues in a protein
anode: the electrode of an electrochemical cell at which oxidation occurs
cathode: the electrode of an electrochemical cell at which reduction occurs