Electrophoresis

Question 1

What is electrophoresis?

Answer 1

• 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)

Question 2

Application of electrophoresis in biosciences

Answer 2

• 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.)

Question 3

History of Electrophoresis

Answer 3

• 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

Question 4

Principles of Electrophoresis: Net Charge

Answer 4

• 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

Question 5

Principles of Electrophoresis: Size/Shape

Answer 5

• 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

Question 6

Principles of Electrophoresis: Field Strength

Answer 6

• 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)

Question 7

Calculating the electrophoretic mobility of a molecule

Answer 7

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

Question 8

Reactions During Electrophoresis

Answer 8

• Electrolysis takes place during electrophoresis

- In practice, the applied electric field is switched off before sample molecules reach the electrodes

Question 9

How is heat generated during electrophoresis?

Answer 9

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

Question 10

Heating problems

Answer 10

• 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)

Question 11

Avoiding Heating Effects

Answer 11

• 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)

Question 12

Supporting media

Answer 12

• 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

Question 13

Cellulose Acetate

Answer 13

• 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

Question 14

Agarose Gel

Answer 14

• 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

Question 15

Polyacrylamide Gel

Answer 15

• 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

Question 16

Separation of DNA Fragments

Answer 16

• 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

Question 17

Combs

Answer 17

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

Question 18

Agarose Gel Electrophoresis

Answer 18

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

Question 19

Agarose Gel Electrophoresis: Running Buffers

Answer 19

• 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

Question 20

Types of running buffers in AGE

Answer 20

• 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

Question 21

Agarose Gel Electrophoresis: Loading Buffer

Answer 21

• 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

Question 22

Loading buffers used in AGE

Answer 22

• 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

Question 23

Agarose Gel Electrophoresis: DNA dyes

Answer 23

• 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

Question 24

Polyacrylamide Gel Electrophoresis (PAGE) of DNA

Answer 24

• Used to separate closely-sized DNA fragments

- Used to resolve small DNA fragments to very high resolution (1bp possible)

Question 25

Electrophoresis of Proteins

Answer 25

- net charge of a protein molecule is pH-dependent due to the presence of several ionisable groups - Size and shape of protein can vary considerable: globular (approximately spherical), fibril (sheets)

Question 26

Ionisable Groups in Proteins

Answer 26

- net charge of a protein molecule is pH-dependent due to presence of several ionisable groups – acids and bases - Ionisation state of acids and bases is pH-dependent

Question 27

Henderson-Hasselbach equation

Answer 27

- 𝑝𝐻=𝑝𝐾_𝑎 + log⁡([𝐴−])/([𝐴𝐻]) | - 𝑝𝐻=𝑝𝐾_𝑎 + log⁡([𝐴])/([𝐴𝐻+])

Question 28

Protein Titration Curves

Answer 28

- differences in charge can be used to separate proteins - pH at which there is no net charge is the isoelectric point (pI) - pI is the resultant of all pKa’s of all ionisable groups in a protein - Each pKa can easily shift a full pH-unit up or down depending on its chemical surroundings, making pKa and resultant pI very difficult to predict

Question 29

PAGE of Proteins: Making the gel

Answer 29

- Polymerisation of acrylamide in presence of bis-acrylamide creates network of polyacrylamide chains crosslinked with bis-acrylamide molecules - Solutions of a mix of acrylamide and bis-acrylamide are sold commercially - Pore-size is determined by the % of acrylamide used in combo with acrylamide:bis-acrylamide ratio (37.5:1 is standard for PAGE of proteins) - Polymerisation is initiated by sulfate radicals formed from a reaction between TEMED and persulfate

Question 30

General principle of PAGE

Answer 30

Discontinuous Gel matrix - Stacking gel: low % acrylamide - wide pore size - Running gel: higher % acrylamide -actual separation - Stacking happens at the interface of stacking-running gel Native PAGE: proteins in their native form - Mobility is complex: shape, size and charge all play a role - Oligomeric state can play a role - Some proteins never enter gel matrix because they're positively charged or neutral

Question 31

Non-dissociating PAGE

Answer 31

- (aka Native PAGE) proteins remain in their normal conformations during electrophoresis - Used when separated proteins must preserve their biological activity (e.g. enzyme activity)

Question 32

Dissociating PAGE

Answer 32

- proteins are denatured and dissociated by treatment with a detergent (typically Sodium Dodecyl Sulfate (SDS), agents that disrupt disulphide bridges (b-mercaptoethanol), and heat (boiling of samples)

Question 33

Proteins in dissociating PAGE

Answer 33

- can be separated according to length of each polypeptide chain - Protein samples are heated in a mix of * SDS (denaturing protein/ensuring separation is based solely on protein size) * b-mercaptoethanol (breaking disulfide bridges to ensure complete denaturation of proteins) * Glycerol (to make sample sink into the loading well) * Bromophenol Blue (a blue dye to make sample visible during loading and running) * Buffer (whichever buffer is used for the stacking gel)

Question 34

Sodium Dodecyl Sulfate PAGE (SDS-PAGE)

Answer 34

- Denaturation with negatively charged detergent that unfolds protein into linear chains with charge directly proportional to protein size - b-mercaptoethanol reduces disulfide bridges to ensure complete unfolding of all proteins - SDS is present in gel, sample and running buffer to ensure denaturation of the proteins - When coloured band (bromophenol blue dye) departs gel in compartment of the +-electrode the run can be stopped - After gel is finished the proteins need to be stained to visualize them

Question 35

Coomassie Blue

Answer 35

- Most widely used stain for protein separations in gels - Detection limit ~0.2ug/band - Staining quantitative up to 20ug for some proteins - Original protocols using R-form involved the use of methanol and acetic acid - Modern protocols use a colloidal form of the G-250 stain: * Dye is suspended in aqueous solution. No use of solvents other than a small % of ethanol (~1% (v/v)) * Phosphoric acid in solution lowers pH to help fix proteins * De-staining not necessary for fast results * Washing to remove residual stain can be done with water

Question 36

Silver staining

Answer 36

- Used for greater sensitivity of staining (ng or even fg protein) - More laborious and expensive than Coomassie blue method: staining protocol takes more time, requires highly pure water to prevent high background staining, staining may be non-specific (DNA and polysaccharides stain too), sensitive to impurities e.g. fingerprints

Question 37

Gradient Polyacrylamide Gels

Answer 37

- %Acrylamide increases (and hence pore size decreases), in direction of protein migration (e.g. 5-20%) - Able to resolve protein mixtures with a wide range of Mr - As proteins migrate into regions of decreasing pore size, the movement of leading edge of a zone will become increasingly restricted - trailing edge of the zone can catch up, resulting in considerable zone sharpening

Question 38

Analysis of Gels

Answer 38

- Dedicated instruments eg. laser densitometers - Gel scanning (absorbance of coomassie blue-stained bands at 560 - 575nm measured) - Most gels are now recorded using a digital camera above a white glass transilluminator * Images (taken with any camera) can be inlayed and band intensity can be quantified (provided you have a reference point) by software

Question 39

Storing Gels

Answer 39

- Gels can be preserved in 7% acetic acid | - Or dried and stored at room temperature using a commercially available gel drier

Question 40

Isoelectric Focusing (IEF)

Answer 40

- carried out using a pH gradient formed using ampholytes (zwitterions, e.g. amino acids with basic (-NH3+) and carboxylic acid group (-COO-) - mixture of ampholytes is placed between anode and cathode - When electric field is applied, each ampholyte migrates to its own isoelectric point (pI) and forms a stable pH gradient - Using polyacrylamide gel as a supporting medium, and a narrow pH gradient, proteins differing in pI by 0.01 units can be separated

Question 41

2D Electrophoresis

Answer 41

- Very high resolution - Most commonly used to separate proteins - First dimension separates proteins by charge (using isoelectric focusing) - Second dimension separates proteins by molecular mass (using denaturing SDS-PAGE) - allows ~1000 proteins to be separated from a single sample

Question 42

2D Electrophoresis – First Dimension

Answer 42

- Normally carried out in polyacrylamide rod gels - ~7 – 24 cm - pH 3 - 10 range - voltage up to 3500V for ~5h - Necessary to remove resolved gel from glass tube by: cracking the glass in a vice, freezing at -20°C, squirting buffer between glass and gel (rimming) - Gels can be stored frozen until required

Question 43

2D Electrophoresis – Second Dimension

Answer 43

- Polyacrylamide slab gel - Size determined by length of rod gel (and apparatus available) - Typically 0.5 – 1.5 mm thick - Cast in situ with 10-16% gradient + stacking gel - Rod gel equilibrated with SDS-PAGE buffer - Loaded between glass plates of the second gel Sealed in place with polyacrylamide or agarose - Well(s) created for markers - Run at 100-200 V

Question 44

2D Electrophoresis – Data Analysis

Answer 44

- Spot patterns are complex - Computer-aided gel scanners required - Simplifies acquisition, storage and processing of data - Also allows quantification of individual proteins - Internal standards (markers) essential - Allows compensation for gel variations - Spot identity obtained from databases

Question 45

2D Electrophoresis – Spot Analysis

Answer 45

- Spots automatically identified - Based on contrast difference with background - Boundaries defined - pI & MW calculated - Image compared to other gels - 3-20 spots used as tiepoints (reference) - Triangle network created to overlay gel image - Each spot automatically compared to surrounding constellation of spots - Matching/unmatching spots highlighted - Annotation of gel now possible

Question 46

Capillary Electrophoresis (CE)

Answer 46

- Combines high resolving power of electrophoresis with speed and versatility of HPLC - Overcomes major problems of electrophoresis in free solution: poor resolution due to convection currents, diffusion - Heat due to electric current is rapidly dissipated due to high surface:area ratio - Very small samples (5-10 nL) can be used - Wide range of biomolecules can be analysed

Question 47

Capillary Electrophoresis Apparatus

Answer 47

- Fused silica capillary coated with polymer - 25-50 µm internal diameter - Gap in polymer allows detection of sample by UV, visible or fluorescent light - Sample loading electrophoretic (using voltage pulse) or displacement (pressure) - Time of detection is characteristic for each molecular species

Question 48

Electro-Osmotic Flow (EOF)

Answer 48

- Due to net negative charge on fused silica surface at pH > 3.0 - Solvent cations flowing towards cathode > attraction of sample anions to anode - So sample cations and anions attracted towards cathode past detector - ↑negative charge on anion, ↑resistance to EOF so ↓mobility

Question 49

Types of Capillary Electrophoresis

Answer 49

- Capillary Zone Electrophoresis (CZE) - Capillary Gel Electrophoresis (GCE) - Capillary Isoelectric Focusing (CIEF) - Micellar Electrokinetic Chromatography (MEKC or MECC)