MCAT Lab Techniques

native-PAGE

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

reverse phase chromatography

same as thin layer chromatography, except a nonpolar card and polar solvent are used

SDS-PAGE

polyacrylamide gel electrophoresis method for proteins using DENATURING conditions

sodium dodecyl sulfate denatures proteins and gives them a uniform charge, which allows them to be separated solely on mass

should not be used when the epitope of interest involves amino acids that are far from each other, as this could prevent the antibody from binding when the protein is denatured

reducing SDS-PAGE

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

isoelectric focusing

based on proteins’ relative contents of acidic and basic residues

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

Southern blot

detection of a specific DNA sequence in a sample

Northern blot

detection of a specific RNA sequence in a sample

Western blot

detection of a specific protein in a sample

chromatography

stationary phase is typically polar, and therefore polar molecules elute slower

mobile phase is typically nonpolar, and therefore 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

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, which gives it higher resolving power

gas chromatography

vaporizes the liquid before separation; molecules are
separated based on polarity and boiling point

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

most nonpolar compound will be the first peak

gel-filtration chromatography

separates molecules by size rather than polarity; smaller molecules enter the porous gel beads allowing them to elute later while larger molecules elute faster because they do not fit in the pores

cation exchange chromatography

proteins are separated by net charge

negative beads used and negative proteins elute first

anion exchange chromatography

proteins are separated by net charge

positive beads used and positive proteins elute first

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

molecules are spotted on the bottom of a sheet coated in polar silica gel, which is placed in a nonpolar liquid; mobile phase travels up the plate using capillary action

nonpolar molecules travel further up than polar molecules

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

vacuum lowers the air pressure, which decreases the temperature 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 boiling point

extraction

combines two immiscible liquids, one of which easily dissolves the compound of interest

organic layer dissolves nonpolar compounds while aqueous layer dissolves polar/hydrogen bonding compounds