Question

Compare and contrast Native -PAGE, SDS-PAGE, size-exclusion chromatography, and affinity chromatography, ion exchange chromatography. How are components separated by these procedures and how are they detected? Explain details. hHow are each of these techniques carried out?

Answer 1

Native-PAGE:

Native-Page is a type of Polyacrylamide gel electrophoresis (PAGE) technique used for separation of biological macromolecules (such as proteins and nucleic acid) in their native form. Macromolecules in their native state preserve the highest order structure and they are separated based on their electrophoretic mobility which depends upon length, conformation and charge on the molecule.

This technique involves preparation of gel of polyacrylamide. After application of sample on this gel, electricity is applied across the gel so that positively charged biomolecules move towards negative electrode and vice versa. Small molecules move faster than large molecules. After analysis visualization of sample is carried out using suitable stain. Molecular weight of biomolecules can also be determined by comparing with suitable markers.

SDS-PAGE:

SDS-PAGE is a type of Polyacrylamide gel electrophoresis (PAGE) technique used for separation of proteins in presence of denaturing agent SDS (sodium dodecyl sulfate). SDS is an anionic detergent which changes proteins from native form to linear form.

SDS-PAGE is carried out similar to that of native-PAGE. Sample is prepared in presence of SDS so that native structure is broken.

Size exclusion chromatography:

Size exclusion chromatography is a chromatographic technique used for separation of molecules in solution by their size and hence molecular weight. It is used for separation of macromolecules such as proteins, nucleic acid or other polymers.

In size exclusion chromatography sample is allowed to pass through the stationary phase. Small sized molecules get trapped in the stationary phase and large molecules pass easily through stationary phase. Thus, large molecules gets eluted first and then small molecules.

Affinity chromatography

Affinity chromatography is a technique used for separation of biochemical mixtures based on highly specific interactions such as between enzyme and substrate or between antigen and antibody or between receptor and ligand. It is used for various applications such as purification of proteins from cell extracts or nucleic acid purification.

Affinity chromatography involves immobilization of desired ligand on solid support. Sample containing desired substrate along with other impurities is allowed to pass through this solid support. The ligands attached to solid support binds to the desired substrates and other impurities are eluted. The substrate is later on separated from solid support.

Ion exchange chromatography

Ion exchange chromatography involves separation of ions based on their affinity to the ion exchanger. It can be either cation exchange chromatography in which positively charged molecules are attracted to negatively charged solid support or anion exchange chromatography in which negatively charged molecules are attracted to positively charged solid support. It is useful for separation of charged biomolecules such as proteins, nucleic acids, amino acids, peptides, etc.

In ion exchange chromatography suitable ion exchanger is used as the solid support. Sample is passed through the ion exchanger so that desired ionic compound binds to the solid support and impurities are eluted out. Desired compound is then eluted from solid support using suitable eluting agent.