Electrophoresis Flashcards
1
Q
What is electrophoresis?
A
- 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)
2
Q
Application of electrophoresis in biosciences
A
- 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.)
3
Q
History of Electrophoresis
A
- 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
4
Q
Principles of Electrophoresis: Net Charge
A
- 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
5
Q
Principles of Electrophoresis: Size/Shape
A
- 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
6
Q
Principles of Electrophoresis: Field Strength
A
- 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)
7
Q
Calculating the electrophoretic mobility of a molecule
A
- 𝜇=(𝐸×𝑞)/𝑟
- 𝜇 = electrophoretic mobility of the molecule
- 𝐸 = electric field strength
- 𝑞 = net charge of molecule
- 𝑟 = radius of the molecule
8
Q
Reactions During Electrophoresis
A
- Electrolysis takes place during electrophoresis
- In practice, the applied electric field is switched off before sample molecules reach the electrodes
9
Q
How is heat generated during electrophoresis?
A
- generated as a result of the power produced during electrophoresis
- (𝑃=𝐼^2×𝑅, in which 𝐼 = current and 𝑅 = resistance)
10
Q
Heating problems
A
- 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)
11
Q
Avoiding Heating Effects
A
- 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)
12
Q
Supporting media
A
- 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
13
Q
Cellulose Acetate
A
- 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
14
Q
Agarose Gel
A
- 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
15
Q
Polyacrylamide Gel
A
- 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
16
Q
Separation of DNA Fragments
A
- 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
17
Q
Combs
A
- Different-sized combs give wells of different size
- Number of wells can be chosen to suit number of samples
- As number increases, volume decreases
18
Q
Agarose Gel Electrophoresis
A
- DNA sample mixed with loading buffer. Ethidium bromide or SybrSafe is present in the gel
- Electrophoresis run typically for ~45 mins at 100V
19
Q
Agarose Gel Electrophoresis: Running Buffers
A
- 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
20
Q
Types of running buffers in AGE
A
- 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
21
Q
Agarose Gel Electrophoresis: Loading Buffer
A
- 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
22
Q
Loading buffers used in AGE
A
- 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
23
Q
Agarose Gel Electrophoresis: DNA dyes
A
- 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
24
Q
Polyacrylamide Gel Electrophoresis (PAGE) of DNA
A
- Used to separate closely-sized DNA fragments
- Used to resolve small DNA fragments to very high resolution (1bp possible)
25
Electrophoresis of Proteins
- 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)
26
Ionisable Groups in Proteins
- 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
27
Henderson-Hasselbach equation
- 𝑝𝐻=𝑝𝐾_𝑎 + log([𝐴−])/([𝐴𝐻])
| - 𝑝𝐻=𝑝𝐾_𝑎 + log([𝐴])/([𝐴𝐻+])
28
Protein Titration Curves
- 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
29
PAGE of Proteins: Making the gel
- 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
30
General principle of PAGE
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
31
Non-dissociating PAGE
- (aka Native PAGE) proteins remain in their normal conformations during electrophoresis
- Used when separated proteins must preserve their biological activity (e.g. enzyme activity)
32
Dissociating PAGE
- 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)
33
Proteins in dissociating PAGE
- 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)
34
Sodium Dodecyl Sulfate PAGE (SDS-PAGE)
- 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
35
Coomassie Blue
- 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
36
Silver staining
- 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
37
Gradient Polyacrylamide Gels
- %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
38
Analysis of Gels
- 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
39
Storing Gels
- Gels can be preserved in 7% acetic acid
| - Or dried and stored at room temperature using a commercially available gel drier
40
Isoelectric Focusing (IEF)
- 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
41
2D Electrophoresis
- 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
42
2D Electrophoresis – First Dimension
- 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
43
2D Electrophoresis – Second Dimension
- 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
44
2D Electrophoresis – Data Analysis
- 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
45
2D Electrophoresis – Spot Analysis
- 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
46
Capillary Electrophoresis (CE)
- 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
47
Capillary Electrophoresis Apparatus
- 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
48
Electro-Osmotic Flow (EOF)
- 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
49
Types of Capillary Electrophoresis
- Capillary Zone Electrophoresis (CZE)
- Capillary Gel Electrophoresis (GCE)
- Capillary Isoelectric Focusing (CIEF)
- Micellar Electrokinetic Chromatography (MEKC or MECC)
