MCAT Biochem - Protein Analysis Techniques

Native PAGE

Proteins are separated based on their native conformation and charge.

SDS-PAGE

Proteins are denatured and separated based on their molecular weight. Proteins migrate toward the anode (positive electrode).

Reducing Conditions SDS-PAGE

The bands visualized under reducing conditions reflect the size of the individual subunits.

Non-Reducing Conditions SDS-PAGE

For proteins that consist of more than one subunit, the band will reflect the molecular weight of the entire protein.

Western blotting

combines protein electrophoresis with antibody binding to identify target proteins with high specificity and sensitivity

ELISA (enzyme-linked immunoassay)

uses antibodies to detect and quantify proteins directly in a liquid sample. the protein of interest binds to a solid surface, and detection is achieved with an enzyme-linked antibody that produces a measurable signal, usually a color change

Southern Blotting

used to detect specific DNA sequences within a complex DNA sample

Northern Blotting

used to detect specific RNA sequences within a complex RNA sample

Chromatography

technique commonly used for separating a mixture of chemical substances into its individual components. The components that adhere more strongly to the stationary phase travel more slowly compared to those with a weaker adhesion

thin layer chromatography

Gradually by capillary action, the solvent started rising up the silica plate, and as you can see the reaction mixture separated into spots with distinct colors

glass column chromatography

the reaction mixture started separating into three distinct bands

Mobile phase or carrier

solvent moving through the column

Stationary phase or adsorbent

substance that stays fixed inside the column

Eluent

fluid entering the column

Eluate

fluid exiting the column (that is collected in flasks)

Elution

the process of washing out a compound through a column using a suitable solvent

Analyte

mixture whose individual components have to be separated and analyzed

normal-phase chromatography

stationary phase is polar (hydrophilic) in nature and our mobile phase is non-polar (hydrophobic) in nature

reverse-phase chromatographic techniques

stationary phase is non-polar while the mobile phase is polar.

Paper chromatography

compound spotted directly on a cellulose paper

Thin layer chromatography (TLC)

glass is coated with thin layer of silica on which is spotted the compound

Liquid column chromatography

glass column is packed with slurry of silica

Size exclusion chromatography

small molecules get trapped in the pores of the stationary phase, while large molecules flow through the gaps between the beads and have very small retention times. So larger molecules come out first. In this type of chromatography there isn’t any interaction, physical or chemical, between the analyte and the stationary phase.

Ion-exchange chromatography

molecules possessing the opposite charge as the resin will bind tightly to the resin, and molecules having the same charge as the resin will flow through the column and elute out first.

Affinity chromatography

if the molecule is a substrate for the enzyme, it will bind tightly to the enzyme and the unbound analytes will pass through in the mobile phase, and elute out of the column, leaving the substrate bound to the enzyme, which can then be detached from the stationary phase and eluted out of the column with an appropriate solvent.

Gas chromatography

samples are volatilized and the molecule with lowest boiling point comes out of the column first. The molecule with the highest boiling point comes out of the column last