Biochemical Separation and Analysis Techniques

Vocabulary flashcards covering biochemical lab techniques including electrophoresis, blotting, sequencing, chromatography, distillation, and PCR.

Gel Electrophoresis

A technique used for the separation of proteins, DNA, or RNA based on size and/or charge using an electrical field and a gel matrix.

Polyacrylamide

The type of gel used in electrophoresis for the separation of proteins and small molecules of DNA and RNA.

Agarose

The type of gel used in electrophoresis for separating larger molecules of DNA ($> 500\,bp$).

Anode

The positively charged electrode at the bottom of the gel electrophoresis setup toward which negatively charged molecules travel.

Cathode

The negatively charged electrode at the top of the gel electrophoresis setup.

Ladder

A collection of molecules of a known size loaded into a gel lane used as a reference to determine the size of the experimental molecules.

Native-PAGE

A polyacrylamide gel electrophoresis method for proteins that occurs under non-denaturing conditions to separate proteins by size while retaining their structure.

SDS-PAGE

A polyacrylamide gel electrophoresis method that separates proteins solely by mass under denaturing conditions by applying a uniform charge-to-mass ratio.

Sodium Dodecyl Sulfate (SDS)

A negatively charged detergent that denatures proteins and binds one SDS for every two amino acids to give all proteins the same charge-to-mass ratio.

Reducing SDS-PAGE

A method identical to SDS-PAGE but with the addition of a reducing agent like $\beta$-mercaptoethanol to reduce disulfide bridges and completely denature the protein.

Isoelectric Focusing

A gel electrophoresis method that separates proteins based on their relative content of acidic and basic residues using a pH gradient.

Isoelectric Point (pI)

The specific pH at which a protein has a net charge of zero and stops migrating through the pH gradient in an isoelectric focusing gel.

Western Blotting

A technique used for the detection of a specific protein in a sample involving SDS-PAGE separation, transfer to a polymer sheet, and exposure to radiolabeled antibodies.

Southern Blotting

A procedure used to detect a specific DNA sequence in a sample involving restriction enzymes, NaOH denaturation into ssDNA, gel electrophoresis, and communication with a $^{32}P$ radiolabeled DNA probe.

Northern Blotting

A procedure used for the detection of a specific RNA sequence that is nearly identical to Southern blotting but omits the use of restriction enzymes and NaOH denaturation.

Sanger Dideoxynucleotide Sequencing

A method used to determine the sequence of nucleotides in a DNA strand by utilizing modified ddNTPs to control the termination of replication.

Dideoxynucleotides (ddNTPs)

Modified nucleotides missing the $OH$ group on the $3'$ carbon, preventing the formation of new $5' \rightarrow 3'$ phosphodiester bonds.

Chromatography

The separation of two or more molecules from a mixture based on their properties using a stationary phase (typically polar) and a mobile phase (typically non-polar).

High-Performance Liquid Chromatography (HPLC)

A type of liquid chromatography that utilizes high pressures to pass the solvent through a finely-ground stationary phase for higher resolving power.

Gas Chromatography

Also known as gas-liquid chromatography, it separates vaporized molecules using an inert gas mobile phase and a liquid or polymer stationary phase.

Gel-Filtration Chromatography

Also known as size-exclusion chromatography, it separates molecules by size where smaller molecules elute later and larger molecules elute faster.

Ion-Exchange Chromatography

A method that separates proteins by their net charge using a column filled with either positively or negatively charged beads.

Cation-Exchange Chromatography

A form of ion-exchange chromatography using negatively charged beads that attract positively charged proteins, allowing negatively charged proteins to elute first.

Anion-Exchange Chromatography

A form of ion-exchange chromatography using positively charged beads that attract negatively charged proteins, allowing positively charged proteins to elute first.

Affinity Chromatography

A method that separates proteins based on their affinity for a specific ligand bound to beads in the column.

Thin-Layer Chromatography (TLC)

A technique using a sheet coated in an adsorbent material like silica where a mobile phase travels up the plate via capillary action to separate molecules based on polarity.

Rf value

In thin-layer chromatography, the ratio of the distance the molecule traveled from the origin to the distance the solvent front traveled.

Simple Distillation

A technique used to separate two molecules from a solution when their boiling points differ by $25^\circ C$ or greater.

Fractional Distillation

A technique used to separate two molecules from a solution when their boiling points differ by less than $25^\circ C$.

Vacuum Distillation

A technique used to separate two molecules with high boiling points to prevent them from changing chemically during the heating process.

Polymerase Chain Reaction (PCR)

A method used to amplify a small quantity of DNA by several orders of magnitude through cycles of denaturation, annealing, and synthesis.

Taq DNA polymerase

A heat-stable enzyme used in PCR to synthesize new complementary DNA strands at $72^\circ C$.