Electrophoresis

• Separation technique based on movement of charged molecules in an electric field • Widely used biotechnique • Can be used to: separate a complex mixture of molecules, confirm homogeneity of isolated biomolecules • Different molecules move at different rates depending on: net charge, size, shape, strength of applied electric field (applied voltage)

• Separation of nucleic acids (DNA and RNA) in molecular biology (research and diagnostics) • Separation of proteins – in bioscience research and clinical diagnostics • Separation of small charged molecules (eg. amino acids, nucleotides, pharmaceuticals etc.)

• Arne Tiselius first separated plasma proteins by moving boundary electrophoresis (1930s) • Detector measures diffraction changes caused by different sample molecules • Poor resolution due to diffusion and convection currents

• Negatively-charged molecules (anions) move towards the anode (+) • Positively-charged molecules (cations) move towards the cathode (-) • Highly-charged molecules move faster than those with less charge

• Smaller molecules tend to move faster than large molecules • Molecule shape also affects mobility: linear DNA vs circular DNA of same number of bp, globular proteins vs fibrous proteins of similar molecular weight • Increase in medium viscosity is stronger for larger molecules

• Electrophoretic mobility (𝜇) increases with increasing field strength (𝐸) until heating effects occur • Charge:mass ratio (charge:density ratio) considers combined influence of net charge (𝑞) and size on mobility (𝜇) • Size of a molecule directly correlates with the radius of a molecule (𝑟)

• 𝜇=(𝐸×𝑞)/𝑟 • 𝜇 = electrophoretic mobility of the molecule • 𝐸 = electric field strength • 𝑞 = net charge of molecule • 𝑟 = radius of the molecule

• Electrolysis takes place during electrophoresis - In practice, the applied electric field is switched off before sample molecules reach the electrodes

• generated as a result of the power produced during electrophoresis • (𝑃=𝐼^2×𝑅, in which 𝐼 = current and 𝑅 = resistance)

• causes convection currents • may lead to zone broadening by increasing rate of diffusion of both sample and buffer ions • Denaturation of sample proteins due to increased temperature (loss of biological activity e.g. with enzymes) • reduces buffer viscosity, leading to decrease in frictional resistance • Electrophoresis run at constant voltage (common) leads to further heat production (Ohm’s Law - as resistance falls, current increases)

• can be avoided by using a power pack that provides constant power • Using ultra low current is not practical, since it leads to long separation times and therefore increased diffusion • In practice, most electrophoresis equipment incorporates a cooling device (e.g. water cooling)

• contains buffer electrolytes and sample is applied in a discrete location or zone • Sample molecules remain in sharp zones as they migrate at different rates during electrophoresis • Once separated, the molecules are fixed and stained to avoid post-electrophoretic diffusion • Supporting media are inert: provide physical support and help to minimise convection • Agarose and polyacrylamide form gels with pores that have a similar size as the sample molecules: additional molecular sieving achieved, movement of larger, molecules restricted by pores, smaller molecule movement unrestricted

• Hydroxyl groups of cellulose acetylated • Less hydrophilic than cellulose (paper): holds less water • Reduced diffusion with increased resolution • Fairly uniform, large pore structure • No molecular sieving for most molecules

• Manufactured from seaweed • Linear polysaccharide consisting of repeating units of galactose and 3,6-anhydrogalactose • Powder dissolved by boiling in electrophoresis buffer – allowed to gel by cooling (H-bonds form) • 0.5% - 3% (w/v) typical - concentration affects pore size, and hence molecular sieving effect • Low – large pores; higher – small pores

• Prepared by cross-linking polymerised chains of acrylamide using N,N’-methylene bis-acrylamide (bis) • Polymerisation initiated by free radicals produced by ammonium persulphate and N,N,N’,N’-tetramethylethylene-diamine (TEMED) • Pore sizes determined by concentration of acrylamide – highly reproducible

• DNA has a uniform net negative charge per unit length due to phosphate groups • So all DNA molecules have the same charge:mass ratio and separate by size (length) • Electrophoresis of DNA commonly involves: agarose gels for routine separation, polyacrylamide gels for higher resolution

• Different-sized combs give wells of different size • Number of wells can be chosen to suit number of samples • As number increases, volume decreases

• DNA sample mixed with loading buffer. Ethidium bromide or SybrSafe is present in the gel • Electrophoresis run typically for ~45 mins at 100V

• Buffers (pH ≈ 8.0-8.5) behave fairly similar, but small difference in wat is optimal for separation of small (TBE is better) vs larger (TAE is better) DNA fragments • EDTA isn’t necessary for electrophoretic action. Chelates Mg2+, an essential co-factor for nucleases that degrade DNA, i.e. EDTA prevents DNA degradation • Boric acid can inhibit several enzymes used in DNA manipulation, i.e. not suitable in case DNA fragments are purified from agarose gel

• 1x TAE (typically made as a 50x stock solution): 40 mM Tris, 20 mM Acetic Acid, 1 mM EDTA • 1x TBE (typically made as a 10x stock solution): 89 mM Tris, 89 mM Boric Acid, 2 mM EDTA

• Dyes have a dual role: making the sample ‘visible’ when pipetting into loading well, tracking progress of the separation • Glycerol increases sample viscosity and as a result it ‘sinks’ to the bottom of the well during loading • EDTA chelates Mg2+, an essential co-factor for nucleases that degrade DNA, i.e. EDTA prevents DNA degradation

• 10 mM Tris-HCl pH 7.6 • 0.03% (w/v) bromophenol blue (300 bp indicator) • 0.03% (w/v) xylene cyanol FF (4000 bp indicator) • 60% (v/v) glycerol • 60 mM EDTA

• Run in the opposite direction in the gel • Both intensely fluorescent when bound to DNA • EtBr - ‘classic’ DNA dye, SYBR Safe and many other new and supposedly safer/superior alternatives are now commercially available • SYBR Safe has certain advantages: can be exited at lower energy wavelengths (blue light), i.e. no need for use of UV-light, which poses a danger to the user and to the DNA sample, less toxic

• Used to separate closely-sized DNA fragments - Used to resolve small DNA fragments to very high resolution (1bp possible)