Biochemical Techniques Cheatsheet

Cheatsheet Content

1. Double-beam spectrophotometer (15 Marks) Principle Measures the intensity of light as a function of wavelength. Uses two beams of light: one passes through the sample, the other through a reference. Ratio of intensities ($I/I_0$) is used to calculate absorbance. Minimizes errors due to fluctuations in lamp intensity or detector sensitivity. Components Light Source: Deuterium lamp (UV), Tungsten-halogen lamp (Visible). Monochromator: Selects specific wavelength (grating or prism). Beam Splitter: Divides light into sample and reference beams. Sample Compartment: Holds cuvettes for sample and reference. Detectors: Photomultiplier tubes (PMT) or photodiodes convert light into electrical signal. Display/Processor: Processes signals and displays results (Absorbance, Transmittance). Working Light from the source passes through the monochromator. The selected wavelength is then split into two beams. One beam passes through the sample cuvette, and the other through a reference cuvette (containing solvent). Detectors measure the transmitted light intensities ($I$ for sample, $I_0$ for reference). The instrument then calculates absorbance ($\text{A} = \log_{10}(I_0/I)$). The double-beam design continuously corrects for variations in the light source and detector response. Applications Quantification of biomolecules (proteins, nucleic acids). Enzyme kinetics studies. Determination of reaction rates. Monitoring cell growth (turbidity). Characterization of chromophores. Advantages High accuracy and precision. Automatic baseline correction. Stability against lamp fluctuations. 2. FRET (Fluorescence Resonance Energy Transfer) (15 Marks) Principle Non-radiative energy transfer from an excited donor fluorophore to an acceptor fluorophore. Occurs when donor and acceptor are in close proximity (typically 1-10 nm) and their emission/excitation spectra overlap. Efficiency of transfer is inversely proportional to the sixth power of the distance ($R^{-6}$) between donor and acceptor. Conditions for FRET Spectral Overlap: Donor emission spectrum must overlap with acceptor excitation spectrum. Proximity: Donor and acceptor must be within the Förster distance ($R_0$, typically 1-10 nm). Orientation: Donor and acceptor dipoles must be favorably oriented. Mechanism When the donor is excited, instead of emitting a photon, it transfers its energy directly to the acceptor. This causes the acceptor to fluoresce (sensitized emission) while donor fluorescence decreases (quenching). Measurement Measure donor fluorescence decrease. Measure acceptor fluorescence increase. Fluorescence lifetime measurements. $$ E = \frac{R_0^6}{R_0^6 + r^6} $$ Where $E$ is FRET efficiency, $R_0$ is Förster distance, $r$ is actual distance. Applications Protein-protein interaction studies. Conformational changes in proteins. DNA hybridization and protein-DNA interactions. Membrane receptor dimerization. Biosensors for intracellular events. Advantages Measures molecular proximity in living cells. Provides dynamic information on interactions. High spatial and temporal resolution. 3. Reverse Phase HPLC (High-Performance Liquid Chromatography) (15 Marks) Principle Separation based on differential partitioning of analytes between a non-polar stationary phase and a polar mobile phase. "Reverse phase" means the stationary phase is non-polar (e.g., C18, C8 alkyl chains) and the mobile phase is polar (e.g., water, methanol, acetonitrile). More non-polar analytes have stronger interactions with the stationary phase and elute later. Components Solvent Reservoir: Holds mobile phase solvents. Pump: Delivers mobile phase at high, constant pressure. Injector: Introduces sample into the mobile phase flow. Column: Contains stationary phase (e.g., silica beads with C18 coating). Detector: UV-Vis, Fluorescence, Mass Spectrometry, Refractive Index. Data System: Records and processes chromatograms. Working The mobile phase (polar) is pumped through the column (non-polar stationary phase). The sample is injected into the mobile phase stream. Analytes in the sample interact with both the mobile phase and stationary phase. Non-polar analytes are retained longer by the non-polar stationary phase, while polar analytes elute faster. A gradient elution (changing mobile phase polarity over time) is often used to improve separation of complex mixtures. The detector then measures the eluting compounds. Applications Separation and purification of peptides, proteins, nucleic acids. Analysis of drugs and metabolites. Environmental monitoring. Food analysis. Quality control in pharmaceuticals. Advantages High resolution and sensitivity. Quantitative analysis. Versatile for a wide range of analytes. Can be coupled with mass spectrometry (LC-MS). 4. RIA (Radioimmunoassay) (15 Marks) Principle Highly sensitive immunoassay technique that uses radioactively labeled antigens to detect specific antibodies or antigens. Based on competitive binding: labeled and unlabeled antigen compete for a limited number of antibody binding sites. Components Antibody: Specific for the antigen of interest. Labeled Antigen: Antigen labeled with a radioisotope (e.g., $^{125}$I, $^3$H). Unlabeled Antigen: Standard antigen or antigen in the sample. Separation Method: To separate antibody-bound antigen from free antigen. Working A fixed amount of antibody and a fixed amount of radioactively labeled antigen are mixed with varying amounts of unlabeled antigen (standards or sample). Labeled and unlabeled antigens compete for the antibody binding sites. The mixture is incubated to allow equilibrium. Bound antigen (antibody-antigen complex) is separated from free antigen (unbound). Common methods include precipitation, solid-phase binding, or secondary antibody. The radioactivity of either the bound or free fraction is measured using a gamma counter (for $^{125}$I) or liquid scintillation counter (for $^3$H). A standard curve is generated by plotting radioactivity vs. concentration of unlabeled antigen. The concentration of antigen in unknown samples is determined from this curve. Standard Curve Example As the concentration of unlabeled antigen increases, the amount of labeled antigen bound to the antibody decreases, leading to lower measured radioactivity in the bound fraction. Applications Measurement of hormones (e.g., insulin, thyroid hormones). Detection of tumor markers. Drug monitoring. Diagnosis of infectious diseases. Detection of specific proteins at very low concentrations. Advantages Extremely high sensitivity (picogram to nanogram levels). High specificity. Quantitative results. Disadvantages Use of radioisotopes requires special handling and disposal. Limited shelf-life of labeled reagents. Potential health hazards. 5. ESI MS / ESI problem (15 Marks) ESI MS (Electrospray Ionization Mass Spectrometry) Principle: Soft ionization technique that produces gas-phase ions directly from liquid solutions, primarily for large, polar, and thermally labile molecules (e.g., proteins, peptides, nucleic acids). Process: Sample solution is sprayed through a fine needle (capillary) at high voltage, creating a fine mist of charged droplets. Solvent evaporates from droplets as they pass through a desolvation zone, increasing charge density. Coulombic repulsion causes droplets to fission into smaller, highly charged droplets. Eventually, individual ions are released from the droplets ("ion evaporation model" or "charged residue model"). Ions are then directed into a mass analyzer. Key Feature: Often produces multiply charged ions, extending the mass range of the mass spectrometer for large biomolecules. Applications: Protein identification and characterization, peptide sequencing, drug discovery, metabolomics. ESI Problems (Challenges and Limitations) Ion Suppression: Description: Co-eluting matrix components or other analytes can compete for charge or space at the droplet surface, reducing the signal intensity of the analyte of interest. Impact: Decreased sensitivity, inaccurate quantification. Mitigation: Improved chromatographic separation, matrix-matched calibration, internal standards, dilution of sample. Matrix Effects: Description: Non-analyte components in the sample (e.g., salts, buffers, detergents) can interfere with the ESI process. Impact: Ion suppression, signal enhancement, adduct formation, altered charge states. Mitigation: Sample cleanup (e.g., SPE), optimization of mobile phase, using more robust ionization sources if applicable. Limited Analyte Concentration Range: Description: ESI works best within a specific concentration range. Too high concentration can lead to charge saturation and ion suppression; too low can be below detection limits. Impact: Non-linear response, poor reproducibility. Mitigation: Proper sample preparation and dilution. Sensitivity to Salt and Non-volatile Buffers: Description: Non-volatile salts and buffers can precipitate in the ESI source, leading to clogging and unstable spray. Impact: Instrument downtime, poor signal. Mitigation: Use volatile buffers (e.g., ammonium acetate, formic acid), desalting steps, careful sample preparation. Charge State Distribution Complexity: Description: ESI often produces a distribution of charge states for a single molecule, which can complicate data interpretation, especially for mixtures. Impact: Requires deconvolution algorithms, can obscure minor components. Mitigation: Optimization of ESI conditions (e.g., pH, solvent) to favor specific charge states, advanced data analysis. 6. SDS-PAGE / Native PAGE (15 Marks) SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) Principle: Separates proteins primarily by molecular weight. Purpose: Denaturing electrophoresis. Sample Preparation: Proteins are denatured by heating with SDS (an anionic detergent) and $\beta$-mercaptoethanol (or DTT). SDS: Binds to proteins, imparting a uniform negative charge-to-mass ratio and denaturing secondary/tertiary structures. $\beta$-mercaptoethanol: Reduces disulfide bonds, breaking multimeric proteins into individual subunits. Gel Matrix: Polyacrylamide gel (formed by polymerization of acrylamide and bis-acrylamide). The pore size of the gel determines the separation range. Electrophoresis: Proteins, now uniformly negatively charged, migrate towards the positive anode. Migration rate is inversely proportional to the logarithm of their molecular weight. Smaller proteins move faster. Detection: Staining with Coomassie Brilliant Blue or silver stain, or Western blotting. Applications: Determine protein molecular weight, assess protein purity, quantify proteins, identify proteins (Western blot), analyze protein expression. Native PAGE (Native Polyacrylamide Gel Electrophoresis) Principle: Separates proteins based on their intrinsic charge, size, and shape (native conformation). Purpose: Non-denaturing electrophoresis. Sample Preparation: Proteins are prepared in a non-denaturing buffer, without SDS or reducing agents. Proteins retain their native structure, biological activity, and charge. Gel Matrix: Polyacrylamide gel, similar to SDS-PAGE but without SDS in the gel or running buffer. Electrophoresis: Proteins migrate based on their net charge (dictated by amino acid composition and pH of buffer), size, and overall 3D shape. Proteins with higher negative charge and smaller size/more compact shape migrate faster. Detection: Staining, or activity assays if the protein retains enzymatic activity within the gel. Applications: Study protein-protein interactions (e.g., oligomerization states). Identify active enzymes. Analyze protein isoforms or post-translational modifications that alter charge. Purify proteins while maintaining activity. Determine isoelectric points (when combined with isoelectric focusing). Comparison Table Feature SDS-PAGE Native PAGE Denaturing? Yes (SDS, heat, reducing agent) No (proteins retain native state) Separation Basis Molecular Weight Charge, Size, Shape SDS Used? Yes (in sample, gel, buffer) No Reducing Agent? Yes (to break disulfide bonds) No Charge Uniformly negative (SDS-bound) Intrinsic charge of protein Protein Activity Lost Retained (can be assayed) Applications MW determination, purity, quantification Protein interactions, enzyme activity, isoforms 7. Blood Glucose Estimation / Enzyme assay (15 Marks) Blood Glucose Estimation (Enzymatic Method) Principle: Enzymatic methods are highly specific and sensitive for measuring glucose in blood. The most common method uses glucose oxidase and peroxidase. Enzymes Involved: Glucose Oxidase (GOD): Catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide ($H_2O_2$). Peroxidase (POD): Catalyzes the oxidation of a chromogenic substrate (e.g., o-toluidine, 4-aminophenazone, phenol) by $H_2O_2$ to produce a colored product. Reaction Steps: Glucose + $O_2$ + $H_2O$ $\xrightarrow{\text{Glucose Oxidase}}$ Gluconic Acid + $H_2O_2$ $H_2O_2$ + Chromogen $\xrightarrow{\text{Peroxidase}}$ Colored Product + $H_2O$ Measurement: The intensity of the colored product is directly proportional to the glucose concentration and is measured spectrophotometrically at a specific wavelength. Procedure Overview: Collect blood sample. Prepare plasma or serum (or use whole blood for point-of-care devices). Add a known volume of sample to a reagent containing GOD, POD, and the chromogen. Incubate for a set time at a specific temperature. Measure absorbance of the colored product using a spectrophotometer. Compare absorbance to a standard curve of known glucose concentrations. Clinical Significance: Diagnosis and monitoring of diabetes mellitus, hypoglycemia, hyperglycemia. General Principles of Enzyme Assay Definition: A method to measure the activity of an enzyme by quantifying the rate at which it converts its substrate into product. Reaction Rate: Enzyme activity is typically expressed as the amount of product formed or substrate consumed per unit time (e.g., $\mu mol/min$). Factors Affecting Enzyme Activity: Substrate concentration. Enzyme concentration. pH. Temperature. Presence of activators or inhibitors. Types of Enzyme Assays: Continuous Assays (Kinetic Assays): Measure the change in substrate or product concentration over time, providing a reaction rate. Often involve a direct spectrophotometric measurement if the substrate or product absorbs light, or a coupled reaction that produces a detectable signal. Example: Measuring NADH oxidation at 340 nm. Discontinuous Assays (Endpoint Assays): The reaction is stopped after a fixed time, and the amount of product formed or substrate consumed is measured. Suitable when the product or substrate is stable after the reaction is quenched. Example: Glucose estimation described above. Common Readout Methods: Spectrophotometry: Most common; measures absorbance change. Fluorometry: Measures fluorescence intensity. Chemiluminescence: Measures light emission. Radiometric: Measures radioactivity of labeled substrate/product. Electrochemical: Measures changes in electrical properties. Standard Curve: For endpoint assays or for quantifying enzyme concentration, a standard curve (e.g., of product concentration vs. signal) is often required. Units of Enzyme Activity: International Unit (IU or U): 1 IU is the amount of enzyme that catalyzes the conversion of 1 $\mu mol$ of substrate per minute under specified conditions. Katal (kat): 1 kat is the amount of enzyme that catalyzes the conversion of 1 $mol$ of substrate per second. 8. Applications of Affinity/Column Chromatography (15 Marks) Affinity Chromatography Principle: Separation based on specific, reversible binding interactions between a target molecule (e.g., protein) and a specific ligand immobilized on a stationary phase. Mechanism: Binding: Sample is applied to the column. Target molecules bind specifically to the immobilized ligand. Unwanted molecules pass through. Washing: Column is washed to remove non-specifically bound molecules. Elution: Target molecules are eluted by changing conditions (e.g., pH, ionic strength, or adding a competitive ligand) that disrupt the specific binding. Regeneration: Column is regenerated for reuse. Common Ligands: Antibodies (for antigens). Enzyme substrates/inhibitors (for enzymes). Lectins (for carbohydrates/glycoproteins). Metal ions (for His-tagged proteins - IMAC). Streptavidin (for biotinylated molecules). Applications: Protein Purification: Highly effective for purifying specific proteins from complex mixtures (e.g., antibody purification using Protein A/G, His-tag protein purification). Immunoprecipitation: Isolation of specific antigens or antibody-antigen complexes. Removal of Contaminants: E.g., removal of albumin from serum. Enzyme Isolation: Purifying specific enzymes using substrate or inhibitor analogs. Study of Biomolecular Interactions: Investigating binding kinetics and affinities. Advantages: High specificity, high resolution, high recovery, significant enrichment in a single step. General Column Chromatography Applications Column chromatography encompasses various techniques where a stationary phase is packed into a column, and a mobile phase passes through it to separate components. Gel Filtration Chromatography (Size Exclusion Chromatography - SEC): Principle: Separates molecules based on size. Larger molecules elute first as they cannot enter pores of the stationary phase beads. Applications: Desalting, buffer exchange, molecular weight determination, separation of protein complexes, aggregate removal. Ion Exchange Chromatography (IEC): Principle: Separates molecules based on their net charge. Stationary phase contains charged groups (anion or cation exchangers). Applications: Protein purification (e.g., separating proteins with different pI values), nucleic acid purification, peptide separation. Hydrophobic Interaction Chromatography (HIC): Principle: Separates proteins based on hydrophobicity. Proteins bind at high salt concentrations and elute as salt concentration decreases. Applications: Purification of proteins, especially those sensitive to denaturing conditions; used after ammonium sulfate precipitation. Hydrophilic Interaction Liquid Chromatography (HILIC): Principle: Separates polar compounds using a polar stationary phase and a mobile phase with high organic content. Applications: Separation of highly polar compounds that are poorly retained in reverse-phase HPLC, e.g., carbohydrates, nucleotides, amino acids. Chromatography in General: Purification: Isolating specific compounds from complex mixtures. Analysis: Identifying and quantifying components in a sample. Process Monitoring: Ensuring quality control in manufacturing. Fractionation: Separating a sample into different fractions for further analysis. 9. Beer-Lambert's Law and Applications (2 Marks) Beer-Lambert's Law States that the absorbance of a solution is directly proportional to the concentration of the absorbing substance and the path length of the light through the solution. Formula: $A = \epsilon bc$ $A$: Absorbance (unitless) $\epsilon$: Molar absorptivity (L $\cdot$ mol$^{-1}$ $\cdot$ cm$^{-1}$) - a constant for a given substance at a specific wavelength. $b$: Path length (cm) - usually 1 cm in standard cuvettes. $c$: Concentration (mol $\cdot$ L$^{-1}$) Limitations: Applies to dilute solutions, monochromatic light, and non-interacting absorbing species. Applications Quantification: Determining the concentration of substances in solution (e.g., proteins, nucleic acids, drugs) by measuring their absorbance. Kinetic Studies: Monitoring reaction rates by observing changes in absorbance over time. Spectrophotometry: Foundation of UV-Vis spectrophotometry. Purity Assessment: Ratios of absorbances at different wavelengths can indicate purity (e.g., $A_{260}/A_{280}$ for nucleic acids/proteins). 10. Shielding, Deshielding, and Chemical Shift (NMR) (2 Marks) Shielding Electrons around a nucleus generate a local magnetic field that opposes the applied external magnetic field ($B_0$). This reduces the effective magnetic field experienced by the nucleus. Nuclei in electron-rich environments (e.g., alkyl protons) are more shielded and resonate at lower frequencies (upfield, smaller $\delta$ ppm values). Deshielding Electronegative atoms or $\pi$-electron systems (e.g., aromatic rings, double bonds) withdraw electron density from a nucleus. This reduces the shielding effect, increasing the effective magnetic field experienced by the nucleus. Nuclei in electron-deficient environments (e.g., protons next to oxygen, nitrogen, halogens, or in aromatic rings) are deshielded and resonate at higher frequencies (downfield, larger $\delta$ ppm values). Chemical Shift ($\delta$) The difference in resonance frequency of a nucleus from a reference standard (e.g., TMS for $^1$H and $^{13}$C NMR). Expressed in parts per million (ppm) to make it independent of the spectrometer's magnetic field strength. $\delta = \frac{(\nu_{sample} - \nu_{reference})}{\nu_{spectrometer}} \times 10^6$ It is a direct measure of the electronic environment of a nucleus, reflecting shielding/deshielding effects. 11. Compare $^1$H and $^{13}$C NMR (2 Marks) Feature $^1$H NMR $^{13}$C NMR Nucleus Proton ($^1$H) Carbon-13 ($^{13}$C) Natural Abundance ~99.98% ~1.1% Sensitivity High (more sensitive) Low (less sensitive, often requires more sample/scans) Chemical Shift Range ~0-15 ppm ~0-220 ppm (much wider) Information Number of distinct proton environments, integration (relative number of protons), splitting (neighboring protons) Number of distinct carbon environments, type of carbon (CH$_3$, CH$_2$, CH, C - often with DEPT) Coupling Homotopic ($^1$H-$^1$H) coupling is common, leads to splitting (n+1 rule) Heterotopic ($^{13}$C-$^1$H) coupling is strong but often decoupled to simplify spectra; $^{13}$C-$^{13}$C coupling is rare due to low natural abundance Interpretation Good for detailed structural elucidation, connectivity via coupling Good for carbon backbone, functional groups, fewer overlaps due to wide range 12. MALDI TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) (2 Marks) Principle: Soft ionization technique used in mass spectrometry to analyze large biomolecules (proteins, peptides, polymers). Mechanism: Sample is mixed with a UV-absorbing matrix and co-crystallized on a target plate. A laser pulse (UV) strikes the crystal, causing the matrix to rapidly vaporize and desorb. The matrix transfers charge to the analyte molecules, creating gas-phase ions (often singly charged). Ions are accelerated into a Time-of-Flight (TOF) mass analyzer. TOF Analyzer: Measures the time it takes for ions to travel a fixed distance. Lighter ions travel faster and reach the detector first. Mass-to-charge ratio ($m/z$) is determined from flight time. Applications: Protein identification (peptide mass fingerprinting), polymer analysis, microbial identification, quality control of synthetic molecules. Advantages: High mass range, high sensitivity, tolerant to salts and buffers, relatively simple spectra (often singly charged ions). 13. mRNA purification (2 Marks) Principle: mRNA (messenger RNA) constitutes a small percentage (1-5%) of total RNA and is characterized by its poly-A tail (a stretch of adenine nucleotides at the 3' end). Method: Affinity chromatography using oligo-dT beads. Cell Lysis: Cells are lysed to release total RNA. Binding: Total RNA is passed over a column or beads containing immobilized oligo-dT sequences (short chains of thymine nucleotides). Hybridization: The poly-A tail of mRNA molecules specifically binds (hybridizes) to the oligo-dT sequences. Other RNA species (rRNA, tRNA) lack poly-A tails and do not bind. Washing: Non-mRNA is washed away. Elution: mRNA is eluted by changing buffer conditions (e.g., lower salt concentration or increased temperature) to disrupt the oligo-dT:poly-A interaction. Applications: cDNA synthesis, gene expression analysis (RT-PCR, RNA-Seq), cloning, in vitro translation. Advantages: Highly specific for mRNA, yields high purity. 14. 2D Electrophoresis (2 Marks) Principle: Separates proteins based on two independent properties, providing much higher resolution than 1D electrophoresis. First Dimension (Isoelectric Focusing - IEF): Proteins are separated based on their isoelectric point (pI), the pH at which their net charge is zero. Proteins migrate in a pH gradient until they reach their pI. Second Dimension (SDS-PAGE): The IEF gel strip is then placed on top of an SDS-PAGE gel. Proteins are separated based on their molecular weight, as in standard SDS-PAGE. Result: A 2D map of proteins, where each spot represents a unique protein with a specific pI and molecular weight. Applications: Proteomics (protein expression profiling), detection of post-translational modifications (which alter pI or MW), identification of disease biomarkers. Advantages: High resolution for complex protein mixtures, can detect subtle changes in proteins. 15. Peptide fragmentation (2 Marks) Principle: In mass spectrometry, peptides are fragmented into smaller ions, and the mass-to-charge ratios of these fragment ions are used to deduce the amino acid sequence. Common Method: Collision-Induced Dissociation (CID) or Higher-energy Collisional Dissociation (HCD). Peptide ions (precursor ions) are isolated and then collided with an inert gas (e.g., helium, argon). This imparts energy, causing the peptide backbone to cleave at specific amide bonds. Types of Fragment Ions: b-ions: Contain the N-terminus. y-ions: Contain the C-terminus. Other ions (a, x, c, z) are also possible. Data Analysis: The mass differences between consecutive b-ions or y-ions correspond to the mass of individual amino acid residues, allowing for sequencing. Applications: De novo peptide sequencing, protein identification (via database searching), identification of post-translational modifications. 16. TLC and column chromatography (2 Marks) Thin Layer Chromatography (TLC) Principle: A simple, rapid, and inexpensive technique for separating mixtures. Stationary phase is a thin layer of adsorbent (e.g., silica gel, alumina) coated on a flat plate. Mobile phase is a solvent or solvent mixture that moves up the plate by capillary action. Separation: Components separate based on differential adsorption to the stationary phase and solubility in the mobile phase. More polar compounds interact strongly with polar stationary phase and move slower (lower $R_f$). $R_f$ Value: Retention factor, $R_f = \frac{\text{distance traveled by spot}}{\text{distance traveled by solvent front}}$. It is characteristic for a compound under specific conditions. Applications: Monitoring reaction progress, checking purity of compounds, identifying compounds by comparing $R_f$ values, optimizing solvent systems for column chromatography. Column Chromatography Principle: A preparative technique for separating larger quantities of mixtures. Stationary phase is packed into a glass or plastic column. Mobile phase flows through the column by gravity or pressure. Separation: Similar to TLC, components separate based on their differential interactions with the stationary and mobile phases. Fractions containing separated compounds are collected. Types: Adsorption (e.g., silica gel), reversed-phase, ion-exchange, gel filtration, affinity (see earlier section for details). Applications: Purification of compounds (e.g., organic synthesis products, natural products), separation of complex mixtures, preparative scale separations. Comparison TLC is analytical, rapid, small scale, and qualitative. Column chromatography is preparative, slower, larger scale, and can be quantitative. 17. Applications of tracers (2 Marks) Definition: A tracer (or label) is a substance (often a radioisotope or stable isotope, fluorescent tag, enzyme, etc.) introduced into a system to track the movement or fate of another substance. Key Properties: Must behave similarly to the substance being traced but be distinguishable. Applications: Metabolic Pathways: Tracing the fate of specific atoms or molecules through biochemical reactions (e.g., using $^{14}$C-glucose to study glycolysis). Drug Metabolism: Tracking drug absorption, distribution, metabolism, and excretion (ADME) using labeled drugs. Diagnostic Imaging: Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography (SPECT) use radioisotopes to visualize physiological processes in vivo. Environmental Studies: Tracking pollutants, water flow, or nutrient cycling. Immunological Assays: Using enzyme-linked or radio-labeled antibodies/antigens in ELISA or RIA. Molecular Biology: Labeling nucleic acids (e.g., $^3$H or $^{32}$P-labeled probes for Southern/Northern blotting) or proteins. 18. PFGE (Pulsed-Field Gel Electrophoresis) (2 Marks) Principle: A specialized type of gel electrophoresis used to separate very large DNA molecules (up to 10 Mb) that cannot be resolved by conventional agarose gel electrophoresis. Mechanism: Instead of a constant electric field, PFGE applies electric fields that periodically change direction (pulses). This forces large DNA molecules to reorient themselves to move through the gel matrix. Larger DNA molecules take longer to reorient and therefore migrate slower than smaller ones. Applications: Epidemiology: Typing bacterial strains for outbreak investigations (e.g., E. coli O157:H7). Genomics: Constructing physical maps of large genomes, studying chromosomal rearrangements. Yeast Genetics: Separating large yeast chromosomes. Food Safety: Identifying sources of foodborne pathogens. Advantages: Ability to resolve extremely large DNA fragments, high discriminatory power for bacterial typing.