MCAT - Lab Techniques (MilesDown)

Native-PAGE

A polyacrylamide gel electrophoresis method for proteins using NON-DENATURING conditions. Proteins keep their native charge and structure so they are separated based on charge and size.

SDS-PAGE

A polyacrylamide gel electrophoresis method for proteins using DENATURING conditions. Sodium Dodecyl Sulfate denatures the proteins and gives the proteins a uniform charge. This allows them to be separated solely on mass, thus, you can estimate the protein's molecular mass.

Reducing SDS-PAGE

Exactly the same as SDS-PAGE, but with the addition of a reducing agent, b-mercaptoethanol, which will reduce the disulfide bridges and result in a completely denatured protein.

Isoelectric Focusing

basis of their relative contents of acidic and basic residues. The gel has a pH gradient and the proteins will migrate through the gel until they reach the pH that matches their isoelectric point. At the pI, the protein has a neutral charge, so it will no longer be attracted to the anode and it will stop migrating.

Blotting techniques

Southern Blotting: Detection of a specific DNA sequence in a sample.

Northern Blotting: Detection of a specific RNA sequence in a sample.

Western Blotting: Detection of a specific PROTEIN in a sample.

SNOW-DROP

Chromatography

Stationary Phase: Typically polar. Polar molecules elute slower.

Mobile Phase: Typically nonpolar. Nonpolar molecules elute faster.

Liquid Chromatography

Silica is used as the stationary phase while toluene or

another nonpolar liquid is used as the mobile phase.

High-Performance Liquid Chromatography

HPLC is a type of liquid chromatography that uses high pressure to pass the solvent phase through a more finely ground stationary phase which increases the interactions between the molecules and the stationary phase. This gives HPLC higher resolving power.

Gas Chromatography

Vaporizes the liquid before separation. Molecules are

separated based on polarity and boiling point. The

stationary phase is a thin layer of material applied to the inside of the column. Typically the polarity of the stationary phase matches that of the solute. The mobile phase is an inert gas.

Gel-Filtration Chromatography

Separates molecules by size rather than polarity. Smaller molecules enter the porous gel beads allowing them to elute later. Larger molecules will elute faster because they do not fit in the pores and will not be slowed down.

Ion Exchange Chromatography

Separates proteins by their net charge. The column is

filled with charged beads, either POS or NEG.

Cation Exchange: NEG beads used, NEG proteins elute 1st.

Anion Exchange: POS beads used, POS proteins elute 1st.

Affinity Chromatography

Separates proteins based on their affinity for a specific

ligand. Beads are bound to a specific ligand and proteins

with a high affinity for that ligand will bind to the beads.

Proteins with a low affinity for the ligand will elute first.

Thin-Layer Chromatography:

Sheet coated in polar silica gel. Molecules are spotted on

the bottom of the sheet. Sheet is placed in a nonpolar

liquid. Mobile phase travels up the plate using capillary

action. Nonpolar molecules have the highest Rf value.

Simple Distillation

Can be used if the boiling points are under 150°C and are at least 25°C apart.

Vacuum Distillation:

Should be used if the boiling points are over 150°C to prevent degradation of the product. The vacuum lowers the air pressure, which decreases the temp the liquid must reach in order to boil.

Fractional Distillation

Should be used if the boiling points are less than 25°C apart

because it allows more refined separation of liquids by BP.

extraction

Combines two immiscible liquids, one of which easily

dissolves the compound of interest.

Nonpolar Layer: Organic layer, dissolves nonpolar compounds.

Polar Layer: Aqueous (water) layer. Dissolves compounds with hydrogen bonding or polarity.