Lab Techniques

Native-PAGE

Separates proteins in their natural, folded state based on size and charge. Good for analyzing complete protein complexes.

SDS-PAGE

Uses a detergent to denature proteins and coat them in a uniform negative charge. Proteins separate solely based on mass (smaller travels faster).

Reducing SDS-PAGE

Adds a reducing agent (like B-mercaptoethanol or DTT) to explicitly break covalent disulfide bonds, separating multi-subunit complexes.

Isoelectric Focusing (IEF)

Separates proteins based on their pI using a pH gradient gel. Proteins migrate until pH = pI, becoming neutral and stopping.

Size-Exclusion Chromatography (Gel Filtration)

Uses porous beads. Large molecules bypass the pores and elute FIRST. Small molecules get trapped in the maze of pores and travel slowly, eluting LAST.

Ion-Exchange Chromatography

Column is coated with a charge. Cation-exchange is negatively charged to trap positive cations (allowing anions to elute first).

Affinity Chromatography

Column is coated with a specific receptor or antibody to trap only the target protein while everything else washes out.

SNOW DROP Mnemonic

Southern Blot = DNA. Northern Blot = RNA. Western Blot = Protein.

Southern Blot

Uses restriction enzymes and radioactive probes to detect specific DNA sequences.

Northern Blot

Assesses specific RNA and gene transcription levels.

Western Blot

Uses specific primary and secondary antibodies to detect and quantify target proteins.

Restriction Endonucleases

Enzymes that cut DNA, typically recognizing palindromic sequences (e.g., 5'-GAATTC-3') to leave sticky or blunt ends.

PCR (Polymerase Chain Reaction)

Massively amplifies a specific DNA sequence. Requires heat-stable Taq Polymerase, DNA primers, and thermocycling.

PCR Thermocycling Steps

Denaturation (~95°C to break H-bonds), Annealing (~55-65°C for primers to attach), Extension (~72°C for Taq to synthesize).

Sanger Sequencing (Dideoxy Sequencing)

Uses specific modified nucleotides called ddNTPs which lack a 3'-OH group. Incorporation completely stops elongation, creating fragments to read the sequence.