Characterization Methods

Northern Blotting

This test detects SPECIFIC RNA SEQUENCES to measure gene expression. RNA is extracted, separated by AGAROSE GEL ELECTROPHORESIS, transferred to a membrane, and hybridized with a LABELED DNA OR RNA PROBE. It is used to determine WHEN AND WHERE GENES ARE TRANSCRIBED. KEY SAMPLE = RNA, distinguishing it from other blots.

Southern Blotting

This technique identifies SPECIFIC DNA SEQUENCES in a sample. Genomic DNA is digested with restriction enzymes, separated by AGAROSE GEL ELECTROPHORESIS, transferred to a membrane, and hybridized with a LABELED DNA PROBE. It’s used for GENE MAPPING, MUTATION DETECTION, AND DIAGNOSTICS. KEY SAMPLE = DNA.

Western Blotting

This test detects SPECIFIC PROTEINS in a sample. Proteins are separated by SDS-PAGE, transferred to a membrane, and probed with PRIMARY ANTIBODIES, followed by labeled secondary antibodies for visualization. It’s used in PROTEIN EXPRESSION ANALYSIS, DISEASE DIAGNOSIS, AND VERIFICATION OF PROTEIN IDENTITY. KEY SAMPLE = PROTEINS.

Eastern Blotting

This test identifies POST-TRANSLATIONAL MODIFICATIONS (like glycosylation) of PROTEINS. Similar to Western blotting, proteins are separated by SDS-PAGE and transferred to a membrane, but are then probed with LIGANDS OR SUBSTRATES specific for the modification, not antibodies. KEY SAMPLE = MODIFIED PROTEINS.

SDS PAGE

SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS separates DENATURED PROTEINS BASED ON MOLECULAR WEIGHT. SDS binds uniformly to proteins, giving them a NEGATIVE CHARGE so they move only according to SIZE THROUGH A POLYACRYLAMIDE MATRIX. Used before Western blotting and for assessing PROTEIN PURITY.

Quantitative Polymerase Chain Reaction (qPCR)

Amplifies and quantifies DNA using fluorescent dyes or probes during the PCR cycles. It’s highly sensitive and used to measure gene expression, detect mutations, or assess viral loads in real time. Requires a known target sequence and a standard curve for absolute quantification.

Reverse Transcriptase Polymerase Chain Reaction

Converts RNA into (complementary) cDNA using reverse transcriptase before amplifying it with PCR. Used to measure gene expression and detect RNA viruses. Unlike qPCR, it’s typically endpoint analysis unless combined with real-time detection.

Column chromatography

Separates compounds based on polarity using a solid stationary phase (typically silica or alumina) and a liquid mobile phase. More polar compounds adhere to the polar stationary phase and elute slower. Used for purifying chemical mixtures, especially in organic synthesis.

Affinity Chromatography

Isolates specific molecules (usually proteins) based on highly selective binding to a ligand attached to the stationary phase. The mobile phase washes away unbound molecules. Common in enzyme purification or antibody isolation due to its high specificity.

Cation exchange Chromatography

Separates positively charged proteins or ions using a negatively charged stationary phase. More positive molecules elute later. It’s used when working with proteins at a pH below their isoelectric point, making them positively charged.

Anion exchange chromatography

Opposite of cation exchange—it separates negatively charged molecules using a positively charged stationary phase. Effective when working with DNA, RNA, or proteins that are negatively charged at a given pH.

Isoelectric point focusing

Separates proteins based on their isoelectric point (pI) in a pH gradient. Each protein migrates to the point in the gel where its net charge is zero. Useful for high-resolution protein separation, especially in 2D gel electrophoresis.

Mass Spectrometry

Identifies molecules by measuring their mass-to-charge ratio (m/z). Molecules are ionized, sorted by mass, and detected. Used for protein identification, metabolomics, and compound verification. Peaks correspond to specific fragments or molecular weights.

UV Spectrometry

Measures absorbance of UV light, typically at 260 nm for nucleic acids and 280 nm for proteins. Used to quantify DNA, RNA, and protein concentrations. Absorbance is directly proportional to concentration

Infrared Spectroscopy

Identifies functional groups in molecules by measuring absorption of IR light, causing bond vibrations. Key peaks: ~1700 cm⁻¹ (C=O), ~3300 cm⁻¹ (O-H or N-H), ~1600 cm⁻¹ (C=C). Used in organic chemistry for structural analysis.

Nuclear Magnetic Resonance Imaging (NMR) or (MRI)

Uses magnetic fields and radio waves to detect the chemical environment of atomic nuclei (commonly ¹H or ¹³C). It provides detailed info on molecular structure, including carbon skeletons and hydrogen bonding.

Thin Layer chromatography

Separates compounds based on polarity on a thin layer of silica (polar stationary phase). The non-polar mobile phase moves compounds by capillary action. Less polar compounds travel farther. Used for qualitative analysis and purity checks.

Gas Chromatography

Separates volatile compounds based on boiling point and polarity. Uses an inert gas (mobile phase) and a coated capillary column (stationary phase). Lower boiling point and less polar compounds elute faster. Useful in forensics and environmental testing.

Size Exclusion Chromatography

Separates molecules by size using porous beads as the stationary phase. Larger molecules elute first because they cannot enter the pores. Used to determine molecular weight or purify proteins and polymers.

Agarose Gel Electrophoresis

Separates nucleic acids (DNA or RNA) by size. DNA is negatively charged and migrates toward the positive electrode through a porous agarose matrix. Smaller fragments move faster. Used in cloning, PCR analysis, and genotyping.

Native Gel Electrophoresis

Separates proteins based on size, shape, and charge without denaturing them. Unlike SDS-PAGE, proteins retain their native structure and function. Useful for studying protein complexes and conformational states.

Edman Degradation

Sequences amino acids in proteins one at a time from the N-terminus using phenylisothiocyanate (PITC). Best for short peptides (<50 amino acids). Replaced by MS for longer proteins but still valuable in sequence validation.

Bradford protein assay

Measures protein concentration using Coomassie Brilliant Blue dye, which binds to arginine and hydrophobic residues, shifting the dye’s absorbance maximum. Simple, fast, and colorimetric, but less accurate for samples with detergents.

Pneumonic SNOW DROP

South → DNA

North → RNA

O means nothing

West → Protein

immunoprecipitation

Isolates specific proteins or protein complexes from a mixture using an antibody-bound bead. Used for studying protein-protein interactions, modifications, or co-factors. Can be coupled with Western blot or MS.

Enzyme Linked ImmunoSorbent Assay (ELISA)

Detects and quantifies proteins, antigens, or antibodies using a capture antibody, enzyme-linked detection, and a color change reaction. High specificity and sensitivity make it useful in diagnostics and immunology.