SDS-PAGE: Principle, Procedure, and Applications

Electrophoresis moves charged molecules through porous gel under electrical field. Three requirements: charged molecules, porous gel matrix, and applied electrical field. Separation is based on charge-to-mass ratio - higher ratio means faster migration. Migration rate depends on electrical field strength, molecular charge, and frictional drag from gel. Molecular size and conformation also affect migration - larger and more extended molecules migrate slower. This principle enables separation of biomolecules with different charge-to-mass ratios.

Electrophoresis is a separation technique using an electric field through a porous gel matrix to separate charged molecules. DNA and RNA carry negative charges due to phosphate groups, while proteins can have positive or negative charges based on amino acid composition. Migration rate depends on net charge, molecular size, shape, electric field strength, and gel pore size. Gel electrophoresis uses a gel matrix (agarose or polyacrylamide) as the separation medium, with gel composition inversely related to porosity.

Gel electrophoresis separates charged molecules based on charge-to-mass ratio. DNA and RNA carry negative charges from phosphodiester bonds, while proteins require artificial charge addition. The anode is positively charged, cathode negatively charged. Migration depends on net charge (directly proportional) and mass (inversely proportional). Electric force (Q × E) balances frictional force (V × F), yielding Q/E = V/F. Two types exist: moving boundary electrophoresis in liquid media with shifting boundaries, and zone electrophoresis in solid gels (agarose/polyacrylamide) with fixed separation zones.

Gel electrophoresis separates charged biomolecules (DNA, RNA, proteins) by applying an electric field through a porous gel matrix, where migration velocity depends on the molecule's net charge, size, and shape; agarose gels with larger pores (100-500 nm) are suitable for larger DNA molecules, while polyacrylamide gels with smaller pores (10-200 nm) provide better resolution for proteins, and the electrophoretic mobility follows the equation V = QE/f, where V is velocity, Q is net charge, E is electric field, and f is frictional coefficient related to size and shape.

Gel electrophoresis separates macromolecules based on size and charge using an electric field applied across a matrix. Molecules migrate from negative to positive electrodes, with shorter molecules moving faster through gel pores (sieving effect). Two main gel types exist: agarose (large pores, suitable for DNA 50-20,000 bp, easy casting, non-toxic) and polyacryamide (smaller pores, high resolving power for proteins 5-2000 kDa and small DNA fragments, requires chemical polymerization). The gel composition and porosity must match the target molecule's size and properties. Agarose forms via thermal setting while polyacryamide polymerizes chemically. Acrylamide is neurotoxic and requires safety precautions.

Protein denaturation is the loss or destruction of a protein's native three-dimensional structure (secondary, tertiary, and quaternary levels), which results in loss of function; this occurs when denaturing agents such as acids/bases (which disrupt electrostatic interactions by altering protonation states), detergents (which disrupt hydrophobic interactions by inserting into protein interiors), heat (which breaks hydrogen bonds), urea (which competes for hydrogen bonding sites), reducing agents (which break disulfide bridges), and heavy metals (which disrupt both electrostatic interactions and disulfide bonds) interfere with the non-covalent and covalent bonds that maintain protein structure.

Protein denaturation is caused by various agents acting through distinct mechanisms. Physical agents include heat (thermal energy disrupts non-covalent bonds), mechanical agitation (breaks hydrogen bonds and ionic interactions), and hydrostatic pressure (forces water into hydrophobic cores). Chemical agents include acids and bases (alter amino acid ionization states), organic solvents (disrupt hydrogen bonding networks), heavy metals (bind to sulfhydryl groups forming insoluble complexes), and detergents (compete with protein folding by binding hydrophobic regions). Each agent targets specific structural features, demonstrating how different environmental conditions can systematically dismantle the multiple types of interactions that stabilize protein tertiary structure.

Protein denaturation is the loss of secondary, tertiary, and quaternary structure while the primary structure remains intact. Denaturation disrupts hydrogen bonds, hydrophobic interactions, and salt bridges. Physical factors like heat disrupt hydrogen bonds and hydrophobic interactions. Chemical factors like urea disrupt hydrogen bonds, while detergents disrupt hydrophobic interactions. Acids and bases disrupt salt bridges. The protein becomes unfolded and loses its biological function. For example, 70% ethanol denatures proteins to kill bacteria, while 95% ethanol only affects surface proteins.

Protein denaturation can be caused by two main categories of agents. Physical agents include heat (such as cooking eggs, where egg white proteins denature and change from clear liquid to opaque solid), ultraviolet radiation, and X-rays. Chemical agents include acids (like those in the stomach that help digest proteins), heavy metal ions (such as lead and zinc), and other chemicals that disrupt protein structure. These agents work by breaking the non-covalent bonds and interactions that maintain the protein's folded structure.

Denaturation is the process where proteins lose their biological activity by disrupting their tertiary and quaternary structures without breaking covalent bonds; this can be caused by six main factors: heat (which disrupts dispersion forces and hydrogen bonds), changes in pH (which affects salt bridges and hydrogen bonds), reducing agents (which break disulfide bonds), detergents (which disrupt hydrophobic interactions), heavy metals (which interfere with both disulfide bonds and ionic interactions), and mechanical agitation (which physically disrupts weak intermolecular forces).

pH is defined as the negative logarithm of hydrogen ion concentration (pH = -log[H+]), while pOH is the negative logarithm of hydroxide ion concentration (pOH = -log[OH-]), with their sum always equal to 14 at 25°C. The ionic product of water (Kw = [H+][OH-]) equals 10^-14 at 25°C, meaning [H+] = 10^(-pH) and [OH-] = 10^(-pOH). Solutions with pH < 7 are acidic, pH = 7 is neutral, and pH > 7 is basic. The Henderson-Hasselbalch equation (pH = pKa + log([salt]/[acid])) describes buffer systems, where pH equals pKa when salt concentration equals acid concentration, pH > pKa when salt concentration exceeds acid concentration, and pH < pKa when salt concentration is less than acid concentration.

This comprehensive chemistry lesson covers three essential concepts: (1) Molarity, defined as moles of solute per liter of solution, with concentration values expressed using scientific notation (e.g., 10^-7 mole per liter); (2) pH, the measure of hydrogen ion strength calculated as the negative logarithm of hydrogen ion concentration (e.g., pH = -log[H+]); (3) Buffer systems, particularly phosphate buffer, which is most important in biological systems because its pKa value (approximately 7.4) closely matches physiological pH, making it highly effective at maintaining pH stability in the human body.

This comprehensive section covers the foundational concepts of acid-base chemistry. An acid donates protons (H+) while a base accepts them. The pH scale is logarithmic (pH = -log[H+]), with pure water at pH 7 being neutral. Solutions with pH < 7 are acidic (lemon juice, coffee), while pH > 7 are basic (hand soap, bleach). The relationship pH + pOH = 14 and [H+][OH-] = 10^-14 governs all aqueous solutions. Two measurement methods are covered: litmus paper (color-changing indicators) and pH probes (voltage measurement requiring calibration). Buffer systems, consisting of weak acids with conjugate bases, resist pH changes through equilibrium. Equilibrium constants Ka and Kb quantify buffer strength, with pKa + pKb = 14 and Ka × Kb = 10^-14. The magnitude of Ka indicates reaction direction.

pH classification: neutral (pH=7), acidic (pH<7), basic (pH>7). Measure using pH paper or meter. Buffer testing: add 10 drops of acid/base and measure pH change—large changes indicate no buffer, small changes indicate buffer presence. Titration calculations: moles titrant = Molarity × Volume (L); moles analyte = moles titrant (1:1 ratio); Molarity = moles/volume (L). Mass-volume percent = (mass solute/g ÷ volume solution/mL) × 100. Example: 0.004185 mol CH3COOH ÷ 0.005 L = 0.837 M; 0.252 g ÷ 5.00 mL × 100 = 5.03% acetic acid.

pH is defined as the negative logarithm of hydrogen ion concentration (pH = -log[H+]), ranging from 0-14 where values below 7 indicate acidic, 7 neutral, and above 7 basic solutions. Water (H2O) has a bent molecular structure with 104.5° bond angle, exists in three states, and has high specific heat capacity with boiling point 100°C and freezing point 0°C due to hydrogen bonding. Water types include distilled (pure H2O), hard (Ca/Mg ions), saline (Na+/Cl- ions), and groundwater. Water undergoes autoionization: 2H2O ⇌ H3O+ + OH-, with Kw = [H+][OH-] = 1×10⁻¹⁴ at 25°C. The relationship pH + pOH = 14 holds. Acids donate H+ ions (strong acids like HCl completely dissociate, weak acids like CH3COOH partially dissociate), while bases accept H+ or donate OH- (strong bases like NaOH completely dissociate, weak bases like NH4OH partially dissociate). Amphoteric substances like water and amino acids can act as both acids and bases. Buffer solutions resist pH changes when acids or bases are added, consisting of weak acids with conjugate bases (e.g., CH3COOH/CH3COONa for pH 4-6) or weak bases with conjugate acids (e.g., NH4OH/NH4Cl for pH 8-10). The Henderson-Hasselbalch equation (pH = pKa + log([A-]/[HA])) calculates buffer pH from acid dissociation constant and concentration ratios.