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 โ 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 (๐) increases with increasing field strength (๐ธ) until heating effects occur
- Charge:mass ratio (charge:density ratio) considers combined influence of net charge (๐) and size on mobility (๐)
- Size of a molecule directly correlates with the radius of a molecule (๐)
7
Q
Calculating the electrophoretic mobility of a molecule
A
- ๐=(๐ธร๐)/๐
- ๐ = electrophoretic mobility of the molecule
- ๐ธ = electric field strength
- ๐ = net charge of molecule
- ๐ = 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
- (๐=๐ผ^2ร๐ , in which ๐ผ = current and ๐ = 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 (Ohmโs 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 โ allowed to gel by cooling (H-bonds form)
- 0.5% - 3% (w/v) typical - concentration affects pore size, and hence molecular sieving effect
- Low โ large pores; higher โ small pores
15
Q
Polyacrylamide Gel
A
- Prepared by cross-linking polymerised chains of acrylamide using N,Nโ-methylene bis-acrylamide (bis)
- Polymerisation initiated by free radicals produced by ammonium persulphate and N,N,Nโ,Nโ-tetramethylethylene-diamine (TEMED)
- Pore sizes determined by concentration of acrylamide โ 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 โ 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 isnโt 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
