MCAT Physical : Purification Techniques
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Example Question #1 : Electrophoresis
A pH gradient is created on an electrophoresis gel. A mixture of charged proteins is run through this gel and is separated. Which of the following is a true statement?
Proteins that are uncharged at physiological pH can migrate through the gel
Proteins are separated based on size in this gel
A protein stops migrating through the gel when its pH equals the pI of the gel
Other macromolecules cannot be separated via this method
Proteins that are uncharged at physiological pH can migrate through the gel
A pH gradient in a gel facilitates the separation of charged species. The proteins in the mixture encounter different pHs as they travel through the gel. The driving force for the proteins is the electric force. Recall that each protein has an isoelectric point; when the protein's environmental pH equals the isoelectric point, the protein will become neutral and stop migrating (it loses the electric force). The physiological pH is about 7.4. Several amino acids are uncharged at this pH; however, these amino acids will become charged when presented in a different pH environment; therefore, these proteins will be charged and be able to migrate through the gel because of the pH gradient.
The protein will stop migrating when the pH of the environment (gel) equals the pI of the protein. Different types of gel electrophoresis are used to separate molecules based on charge and size. A pH gradient is used to separate molecules based on charge, whereas SDS-PAGE is used to separate molecules based on size. All types of macromolecules (nucleic acids, proteins, lipids, and carbohydrates) can be separated using gel electrophoresis.
Example Question #1 : Electrophoresis
Which of the following is true regarding sodium dodecyl sulfate (SDS)?
It adds positive charge to proteins
It allows for separation of proteins by size
It preserves the mass-to-charge ratio of proteins
It binds to the primary structure of proteins
It allows for separation of proteins by size
SDS is a substance added to polyacrylamide gels to separate substances based on size. This method is called SDS-PAGE. SDS binds to the secondary structure of proteins and gives them an overall negative charge; the bigger the substance the bigger the negative charge. The larger mass compensates for the bigger charge; therefore, SDS makes it so that the mass-to-charge ratio of all substances is the same. This standardizes the electric force experienced by each molecule, making size the only driving force for migration through the gel.
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