Biochemical Laboratory Techniques

Chromatography

Chromatography separates molecules based on differential partitioning between a mobile phase (liquid or gas flowing through the system) and a stationary phase (solid matrix or liquid coating). The separation principle defines the technique. Ion-exchange chromatography separates by net charge: a column packed with charged resin beads (cation-exchange uses negatively charged beads to bind positive proteins; anion-exchange uses positively charged beads to bind negative proteins). Proteins are eluted by increasing salt concentration or changing pH—bound proteins with weaker charge interactions elute first. Size-exclusion (gel-filtration) chromatography separates by molecular size: porous beads trap small molecules in their internal cavities, delaying their passage, while large molecules pass around the beads and elute first. This technique can also estimate molecular weight by comparing elution volume to standards. Affinity chromatography exploits specific binding interactions: the stationary phase is covalently linked to a ligand (e.g., antibody, substrate analog, metal ion for His-tagged proteins), and only the target protein binds while everything else washes through. The bound protein is eluted by adding excess free ligand, changing pH, or adding a competing molecule. High-performance liquid chromatography (HPLC) uses high-pressure pumps to force the mobile phase through a column packed with very fine particles, achieving high resolution and speed. Reverse-phase HPLC uses a nonpolar stationary phase and a polar mobile phase—hydrophobic molecules are retained longer. Thin-layer chromatography (TLC) uses a glass plate coated with silica gel (polar stationary phase); the mobile phase (organic solvent) moves by capillary action. Separation depends on polarity—more polar compounds interact more with the silica and migrate less. TLC Rf = distance traveled by compound / distance traveled by solvent front. Gas chromatography (GC) vaporizes the sample and uses an inert gas as the mobile phase; separation depends on boiling point and polarity of interaction with the column coating. GC is coupled to mass spectrometry (GC-MS) for compound identification.

Electrophoresis

Electrophoresis separates charged molecules by migration through a gel matrix under an applied electric field. The rate of migration depends on charge, size, and shape. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is the most common protein electrophoresis method: SDS, an anionic detergent, denatures proteins and coats them with uniform negative charge (~1 SDS per 2 amino acids), so migration depends solely on molecular weight—smaller proteins move faster through the polyacrylamide mesh. The gel consists of a stacking gel (low % acrylamide, pH 6.8) that concentrates proteins into sharp bands, and a resolving gel (higher % acrylamide, pH 8.8) that separates by size. Reducing agents (β-mercaptoethanol or DTT) break disulfide bonds. A molecular weight ladder (pre-stained standards) runs alongside samples for size estimation. Native PAGE omits SDS and reducing agents, preserving native protein conformation and subunit interactions—separation depends on native charge, size, and shape. Isoelectric focusing (IEF) separates proteins by pI in a pH gradient gel: proteins migrate until they reach the pH equal to their pI, where net charge is zero and migration stops. IEF is the first dimension of 2D gel electrophoresis; SDS-PAGE is the second dimension, resolving first by pI then by molecular weight. Capillary electrophoresis uses a narrow capillary with high voltage for rapid, high-resolution separation. Electrophoresis of DNA uses agarose gels (larger pores than polyacrylamide); DNA is negatively charged from its phosphate backbone and migrates toward the anode—smaller fragments move faster. Ethidium bromide or SYBR Safe intercalates between base pairs and fluoresces under UV for visualization.

Centrifugation

Centrifugation separates particles by sedimentation rate under centrifugal force, which is proportional to particle mass, density, and shape. Differential centrifugation is the simplest form: successive spins at increasing speeds pellet progressively smaller particles. A typical subcellular fractionation protocol: 600 × g for 10 min pellets nuclei and large debris; 15,000 × g for 10 min pellets mitochondria, lysosomes, and peroxisomes; 100,000 × g for 60 min pellets microsomes (ER fragments) and plasma membrane; the final supernatant is cytosol. Density gradient centrifugation separates by buoyant density, not just size. In rate-zonal centrifugation, sample is layered on top of a pre-formed density gradient (e.g., 5–20% sucrose) and centrifuged briefly—particles separate by sedimentation rate (size) and stop when the run ends (before reaching equilibrium). In isopycnic (equilibrium) centrifugation, the sample is mixed with a dense salt (CsCl) or layered on a gradient and centrifuged until each particle reaches the position where its buoyant density equals the surrounding medium density—particles band at their equilibrium position regardless of time. The Svedberg unit (S) measures sedimentation coefficient: 1 S = 10⁻¹³ seconds. Ribosomal subunits are named by their S values (e.g., bacterial 70S ribosome composed of 50S + 30S; eukaryotic 80S = 60S + 40S). Larger S = faster sedimentation, but S values are not additive (they depend on shape as well as mass).

Spectroscopy

Spectroscopy measures the interaction of molecules with electromagnetic radiation. UV-Visible absorption spectroscopy quantifies molecules by their absorbance at characteristic wavelengths, governed by the Beer-Lambert law: A = εcl, where A is absorbance, ε is the molar extinction coefficient (M⁻¹cm⁻¹), c is concentration (M), and l is path length (cm). A = log(I₀/I) = −log(T) where T is transmittance. Proteins absorb at 280 nm due to aromatic residues (Trp ε ≈ 5600, Tyr ε ≈ 1400 M⁻¹cm⁻¹); nucleic acids absorb at 260 nm from their conjugated base rings. The A260/A280 ratio assesses nucleic acid purity: pure DNA ≈ 1.8, pure RNA ≈ 2.0; protein contamination lowers the ratio. Bradford and Lowry assays use colorimetric reactions to quantify protein concentration by absorbance. Fluorescence spectroscopy measures emitted light at a longer wavelength after excitation—intrinsically fluorescent amino acids (Trp, Tyr, Phe) provide structural information; extrinsic fluorophores (GFP, fluorescein) enable sensitive detection in assays. Circular dichroism (CD) spectroscopy measures differential absorption of left- and right-circularly polarized light in the far-UV (190–250 nm) to estimate protein secondary structure content (α-helix, β-sheet, random coil) and in the near-UV (250–320 nm) to probe tertiary structure around aromatic residues. Mass spectrometry measures mass-to-charge ratio (m/z) of ionized molecules with extreme precision. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are soft ionization methods that keep proteins intact. Tandem MS (MS/MS) fragments peptides for sequencing. NMR spectroscopy exploits nuclear spin transitions in a strong magnetic field, providing atomic-resolution structural information in solution—but it requires milligram quantities and is limited to proteins under ~30–40 kDa for full structure determination.

ELISA

Enzyme-linked immunosorbent assay (ELISA) uses antibody-antigen binding specificity coupled with an enzymatic colorimetric reaction for detection and quantification. The four ELISA formats differ in how the antibody-antigen sandwich is assembled. Direct ELISA: antigen is adsorbed to the plate well, then a primary antibody conjugated to an enzyme binds the antigen, and substrate is added to produce a colored product proportional to antigen amount. Simple but low signal amplification. Indirect ELISA: antigen is adsorbed, then an unlabeled primary antibody binds, followed by an enzyme-linked secondary antibody that recognizes the primary antibody. The secondary antibody amplifies the signal (multiple secondary antibodies can bind one primary) and provides flexibility (one enzyme-linked secondary can be used with many different primaries). Sandwich ELISA: a capture antibody is coated on the plate, antigen binds, then a detection antibody (enzyme-linked, recognizing a different epitope) is added. Most specific—two antibodies must bind for signal. Used when antigen is present in a complex mixture (e.g., serum). Competitive ELISA: unlabeled antigen in the sample competes with a fixed amount of enzyme-labeled antigen for a limited number of antibody binding sites. Signal is inversely proportional to sample antigen concentration (more sample antigen = less labeled antigen bound = lower signal). The enzyme is typically horseradish peroxidase (HRP) or alkaline phosphatase (AP), which convert a chromogenic substrate (e.g., TMB for HRP, pNPP for AP) into a colored product measured by absorbance in a plate reader. ELISA is quantitative—comparison to a standard curve of known antigen concentrations yields absolute quantification.

Blotting

Blotting techniques transfer separated molecules from a gel onto a membrane for specific detection. Southern blotting detects DNA: genomic DNA is digested with restriction enzymes, separated by agarose gel electrophoresis, transferred to a nitrocellulose or nylon membrane, and hybridized with a labeled complementary DNA probe. Named after Edwin Southern. Northern blotting detects RNA: total or poly-A RNA is separated on a denaturing agarose gel (formaldehyde to prevent secondary structure), transferred to a membrane, and hybridized with a labeled DNA or RNA probe. Measures transcript size and abundance. Western blotting (immunoblotting) detects proteins: proteins are separated by SDS-PAGE, transferred (electroblotted) to a PVDF or nitrocellulose membrane, blocked with inert protein (BSA or nonfat milk) to prevent nonspecific binding, incubated with a primary antibody against the target protein, then an enzyme-linked or fluorescent secondary antibody, and visualized by chemiluminescence or fluorescence. Western blots provide molecular weight information (band position vs. ladder) and relative abundance (band intensity). They confirm the presence of a specific protein in a complex mixture and can detect post-translational modifications (e.g., a phosphorylation-specific antibody reveals a band shift). Southwestern blotting detects DNA-binding proteins: separated proteins are transferred to a membrane and probed with a labeled DNA sequence—a protein that binds the DNA will retain the label. Loading controls (e.g., β-actin, GAPDH, tubulin) are essential for Western blot normalization to confirm equal loading across lanes. The key distinction: Southern = DNA, Northern = RNA, Western = protein, Southwestern = DNA-protein interaction.

PCR and Sequencing

Polymerase chain reaction (PCR) amplifies a specific DNA sequence exponentially using thermal cycling. Each cycle has three steps: denaturation (~95°C) separates dsDNA into single strands; annealing (~50–65°C) allows primers to hybridize to complementary sequences flanking the target; extension (~72°C) uses Taq polymerase (a thermostable DNA polymerase from Thermus aquaticus) to synthesize new DNA from the primers. Each cycle doubles the target DNA—after n cycles, theoretical yield is 2^n copies. Key components: template DNA, forward and reverse primers (oligonucleotides ~18–25 bp), dNTPs (dATP, dTTP, dCTP, dGTP), Taq polymerase, Mg²⁺ (cofactor), and buffer. RT-PCR (reverse transcription PCR) first uses reverse transcriptase to convert RNA to cDNA, then amplifies the cDNA by PCR—used to study gene expression from mRNA. Quantitative real-time PCR (qPCR) monitors amplification in real time using fluorescent reporters (SYBR Green, which intercalates into dsDNA, or sequence-specific TaqMan probes). The Ct (cycle threshold) value—the cycle at which fluorescence crosses a detection threshold—is inversely proportional to the initial template amount. Lower Ct = more starting template. RT-qPCR combines reverse transcription with qPCR for quantifying mRNA levels. DNA sequencing: Sanger (chain-termination) sequencing uses dideoxynucleotides (ddNTPs), which lack a 3′-OH group and terminate DNA synthesis upon incorporation. Four separate reactions (one for each ddNTP) generate fragments of different lengths that are separated by capillary electrophoresis; the terminating ddNTP at each position reveals the sequence. Next-generation sequencing (NGS) parallelizes millions of sequencing reactions: Illumina uses sequencing-by-synthesis with reversible fluorescent terminators; nanopore sequencing threads DNA through a protein pore and detects current changes as each base passes. For the MCAT, focus on the logic of each technique—PCR amplifies, qPCR quantifies, Sanger terminates with ddNTPs, and NGS parallelizes.

Purification and Assay Logic

Protein purification follows a logical sequence: (1) source selection (tissue, cell culture, recombinant expression in E. coli, yeast, or mammalian cells), (2) lysis to release cellular contents (sonication, French press, detergent, or enzymatic lysis), (3) clarification by centrifugation to remove debris, (4) sequential fractionation by solubility (ammonium sulfate precipitation—proteins precipitate at different salt concentrations), (5) chromatographic separation (often ion-exchange followed by size-exclusion or affinity), and (6) analysis of purity and activity at each step. A purification table tracks total protein, total activity, specific activity (activity/mg protein), yield (% activity recovered), and purification factor (fold increase in specific activity). Specific activity increases as contaminating proteins are removed, reaching a maximum plateau when the protein is pure. SDS-PAGE confirms purity (a single band). Salting out exploits the Hofmeister series: high salt concentrations compete with proteins for water of hydration, reducing solubility and causing precipitation. Ammonium sulfate is commonly used because it is highly soluble, cheap, and does not denature most proteins. Dialysis removes small molecules (salts, cofactors) by diffusion across a semipermeable membrane—proteins are retained inside the dialysis bag while small solutes equilibrate with the external buffer. Ultrafiltration uses pressure and a membrane with a defined molecular weight cutoff to concentrate proteins and exchange buffers. Activity assays must be specific to the protein of interest and preferably continuous (spectrophotometric). Common assay strategies: measure consumption of a chromogenic substrate (e.g., NADH at 340 nm), production of a colored product, or coupling the reaction to an indicator enzyme. The logic unifying all purification is: exploit a unique property of your protein (charge, size, hydrophobicity, binding specificity) to separate it from everything else, and measure its activity at every step to confirm you still have functional protein.

How it works

Biochemical techniques share a fundamental logic: separate, detect, quantify. Chromatography partitions molecules between mobile and stationary phases based on charge (ion-exchange), size (gel-filtration), or binding (affinity). Electrophoresis uses an electric field to drive charged molecules through a sieving matrix—SDS equalizes charge so size alone determines migration rate. Centrifugation exploits differences in mass, density, and shape to pellet or band particles. Spectroscopy identifies molecules by their characteristic absorption or emission of light, with Beer-Lambert converting absorbance to concentration. ELISA and blotting harness the exquisite specificity of antibodies to pick one protein out of thousands, amplifying the signal through enzyme-linked detection. PCR exponentially amplifies a DNA sequence defined by two primers; qPCR measures that amplification in real time to quantify starting template. Every technique connects a molecular property to a measurable output—the MCAT tests your ability to trace that connection and interpret the resulting data.

How it works

Biochemical techniques share a fundamental logic: separate, detect, quantify. Chromatography partitions molecules between mobile and stationary phases based on charge (ion-exchange), size (gel-filtration), or binding (affinity). Electrophoresis uses an electric field to drive charged molecules through a sieving matrix—SDS equalizes charge so size alone determines migration rate. Centrifugation exploits differences in mass, density, and shape to pellet or band particles. Spectroscopy identifies molecules by their characteristic absorption or emission of light, with Beer-Lambert converting absorbance to concentration. ELISA and blotting harness the exquisite specificity of antibodies to pick one protein out of thousands, amplifying the signal through enzyme-linked detection. PCR exponentially amplifies a DNA sequence defined by two primers; qPCR measures that amplification in real time to quantify starting template. Every technique connects a molecular property to a measurable output—the MCAT tests your ability to trace that connection and interpret the resulting data.

Comparisons

• B/B (SDS-PAGE and Western blot): The most common lab technique passages. You must determine molecular weight from band position, confirm protein expression (band present/absent after knockdown or induction), and interpret loading controls.
• B/B (PCR and RT-qPCR): Given Ct values, determine relative gene expression. Lower Ct = higher expression. A ΔCt of 1 corresponds to ~2-fold difference. Understand the difference between detecting DNA (PCR) and measuring mRNA expression (RT-qPCR).
• B/B (Chromatography logic): Predict elution order from protein properties. In cation-exchange at pH 7, positively charged proteins bind and elute last. In size-exclusion, large proteins elute first. In affinity chromatography, only the target is retained.
• B/B (ELISA interpretation): Sandwich ELISA signal ∝ antigen concentration. Competitive ELISA signal ∝ 1/antigen concentration. Indirect ELISA tests for the presence of antibodies in patient serum (e.g., HIV testing).
• B/B (Blotting nomenclature): Southern = DNA, Northern = RNA, Western = protein. This appears as a standalone discrete question—don't mix them up.
• C/P (Spectroscopy): Beer-Lambert law A = εcl. Absorbance is linear with concentration up to ~1.0; above that, deviations occur. A260/A280 ratios for nucleic acid purity assessment are standard lab math.

Common confusions

• Confusing Southern, Northern, and Western blots. Southern = DNA (named after Edwin Southern). Northern = RNA (play on Southern). Western = protein. Southwestern = DNA-protein interaction. This is pure memorization.
• Assuming SDS-PAGE separates only by charge. SDS denatures proteins and coats them with uniform negative charge—separation is by molecular weight ONLY. But remember that in the absence of reducing agent, disulfide-linked subunits remain connected and run as a larger complex.
• Forgetting that smaller DNA fragments migrate faster (farther) in agarose gels. The smallest bands are at the bottom of the gel, closest to the anode.
• Misidentifying the elution order in size-exclusion chromatography. Large molecules elute FIRST because they are excluded from the porous beads. Small molecules enter the beads' pores and take a longer path through the column. This is counterintuitive—think 'big can't fit in, so goes around.'
• Confusing isoelectric focusing with SDS-PAGE. IEF separates by pI (pH gradient gel, no SDS). SDS-PAGE separates by molecular weight. 2D electrophoresis is IEF first, then SDS-PAGE perpendicularly.
• Misinterpreting qPCR Ct values. Lower Ct means MORE starting template (fluorescence crosses threshold earlier). A ΔΔCt calculation is used for relative quantification, but the MCAT typically stops at the conceptual level.
• Treating PCR cycle number as linearly related to product. Amplification is exponential (2^n). After 30 cycles, a single target molecule yields ~10^9 copies in theory.
• Forgetting that ddNTPs lack BOTH a 2′-OH and a 3′-OH. The missing 3′-OH prevents phosphodiester bond formation with the next nucleotide, terminating chain elongation. This is the basis of Sanger sequencing.
• Confusing Bradford and Lowry assays. Both are colorimetric protein assays but with different chemistries. Bradford uses Coomassie dye binding; Lowry uses copper reduction and Folin reagent. The MCAT is more likely to ask about the concept of using a standard curve (BSA) than the chemistry.
• Not recognizing that a purification table tracks specific activity, not total activity. Specific activity = activity/mg protein, which should INCREASE with each purification step. Total activity may decrease due to losses.

Quick review

• Chromatography: ion-exchange (charge), size-exclusion (size—large elute first), affinity (specific binding), HPLC (high pressure, fine particles), TLC (polarity, Rf = distance ratio), GC (volatile compounds, boiling point).
• SDS-PAGE: SDS denatures + coats with uniform negative charge → separates by molecular weight only. Smaller = faster = farther. Reducing agent (βME/DTT) breaks disulfide bonds.
• Native PAGE: no SDS, no reducing agent → proteins remain folded; separation depends on native charge, size, shape.
• Isoelectric focusing (IEF): pH gradient gel, proteins migrate to pH = pI, stop when net charge = 0.
• 2D electrophoresis: IEF (1st dimension, pI) + SDS-PAGE (2nd dimension, MW).
• Centrifugation: differential (sequential speeds pellet smaller particles), density gradient (rate-zonal by size, isopycnic by density). Svedberg (S) = sedimentation coefficient; 70S = 50S + 30S (not additive).
• Beer-Lambert: A = εcl. Proteins at 280 nm (Trp, Tyr), DNA/RNA at 260 nm. A260/A280: pure DNA ~1.8, pure RNA ~2.0.
• ELISA: direct (enzyme-linked 1° Ab), indirect (enzyme-linked 2° Ab, more signal), sandwich (capture + detection Abs, most specific), competitive (signal inversely ∝ Ag).
• Blotting: Southern = DNA, Northern = RNA, Western = protein. All: gel → membrane → probe (labeled DNA/RNA/antibody). Western loading control (β-actin, GAPDH) confirms equal loading.
• PCR: template + primers (flank target) + dNTPs + Taq polymerase. Cycles: denature (95°C), anneal (50–65°C), extend (72°C). Exponential amplification: 2^n copies after n cycles.
• RT-PCR: RNA → cDNA (reverse transcriptase) → PCR. qPCR: fluorescence in real time; Ct = cycle at detection threshold; lower Ct = more template.
• Sanger sequencing: ddNTPs (lack 3′-OH) terminate chain extension; fragments separated by capillary electrophoresis; sequence read from terminating ddNTP.
• Purification logic: lysis → centrifugation (clarify) → ammonium sulfate precipitation → chromatography (IEX then SEC or affinity) → dialysis/ultrafiltration. Specific activity (U/mg) increases with purity.