ELECTROPHORESIS Images
DEFINITION Electrophoresis is a procedure for separating a mixture of charged molecules through a stationary material (gel) in an electrical field. It is a powerful tool for separating amino acids, peptides, proteins and nucleic acids
Theory
Ampholyte amino acid Zwitter ion R-CH.COO - R-CH.COO- R-CH.COOH NH 2 NH 3 + NH 3 + anion alkaline IEP acidic cation ph ph
Factors affecting the velocity of migration (v) of molecules in an electric field 1- Electric field strength (E). 2-Net charge on the molecule (z). 3- Friction coefficient (f) or viscous drag. f depends on :a- mass and shape of migrating molecule,and, b-viscosity of the separating medium. Radius of v= Ez f (f= r n k) migrating molecule viscosity of constant the separating medium.
Factors affecting the velocity of migration (v) of molecules in an electric field (continued) 4- Temperature. 5- Buffers( see later). 6- Wick flow and electroendosmosis. ( see later).
Electrophoresis Equipment Power supply Electrophoresis unit: for the separation of the required molecules. In addition a system is needed to : a- visualize separated molecules. b- quantitate separated molecules (viz: densitometer or colirometer).
Power supply Constant Voltage OR Constant Current Current Heat= E I t provides a relatively constant rate of migration (time in seconds) EMF (volts) current (amperes)
Heat resistance V= IR V constant= I R migration rate V = I constant R heat effect is decreased
Electrophoresis unit 1- Two electrodes : cathode- and anode+ 2-Buffer reservoirs 3- A support for the electrophoresis medium. 4- An insulating cover to minimize evaporation of buffer.
Buffers Importance: A- Transmit current. B- Adjust ph determine charge on solute C- Facilitate the migration of substances to be separated. **The rate of migration of substances depends upon : i- ph ii- ionic strength (high give sharp bands but has drawbacks- mention) iii- composition of buffer:eg Tris acetate, Trisborate, Tris-borate-EDTA(TBE), citrate..etc
Buffers Small volumes electrolysis discarded Large volumes can be good culture medium for bacteria should be refrigerated at 4 o C
Supporting medium. Cellulose acetate membrane (made by treating cellulose with acetic anhydride). Gels (most common). a- Agarose b- Polyacrylamide (PAG). They act also as molecular sieves and so separate according to M.W. in addition to charge.
The actual process of DNA electrophoresis
Example DNA Electrophoresis
The instrument being used to load the samples is called a Micropipettor and can pipette very small volumes
Once the gel has been prepared and loaded, the electrical leads on the running tank are connected to a power supply like the one shown in the photo. The power supply has 3 needle gauges on it, showing what the voltage, current, and power levels
The box the gel is sitting on is called a UV Transilluminator, wear a UVprotective face shield.
Organic molecules such as DNA are charged. DNA is negatively charged because the phosphates (red circles) that form the sugar-phosphate backbone of a DNA.molecule have a negative charge
DNA. The gel is a jello-like material, usually agarose, a substance derived from seaweed. Holes are created in the gel. These will serve as a reservoir to hold the DNA solution.
DNA solutions (mixtures of different sizes of DNA fragments) are loaded in a well in the gel.
The gel matrix acts as a sieve for DNA molecules. Large molecules have difficulty getting through the holes in the matrix. Small molecules move easily through the holes Because of this, large fragments will lag behind small fragments as DNAs migrate through the gel.
As the separation process continues, the separation between the larger and smaller fragments increases
The box the gel is sitting on is called a UV Transilluminator, wear a UVprotective face shield.
Molecular weight markers are often electrophoresed with DNAs. Molecular weight markers are usually a mixture of DNAs with known molecular weights Molecular weight markers are used to estimate the sizes of DNA fragments in your DNA sample
The actual process of DNA electrophoresis is again shown in the next few slides.the first step is to prepare a tray to hold the gel matrix (agarose).the ends of the tray are taped.
.A "gel comb" is used to create holes in the gel
.The gel comb is placed in the tray
Agarose powder is mixed with a buffer solution, usually tris borate EDTA (TBE buffer). The solution is heated until the agarose is dissolved. The hot agarose solution is poured into the tray and allowed to cool.
After the gel is cooled, tape is removed from the ends of the gel tray and the gel tray is placed in an electrophoresis chamber. The electrophoresis chamber is filled with buffer, covering the gel. This allows electrical current from poles at either end of the gel to flow through the gel. Finally, DNA samples are mixed with a "loading dye". The loading dye allows you to see the DNA as you load it and contains glycerol or sucrose to make the DNA sample heavy so that it will sink to the bottom of the well.
A safety cover is placed over the gel and electrodes are attached to a power supply. Electrical current is applied. DNA fragments will migrate through the gel at various rates, depending on their size. When the dye marker indicates that DNA fragments have moved through the gel, the current is turned off and the gel is removed from the tray.
DNAs are visualized by staining the gel with ethidium bromide which binds to DNA and will fluoresce in UV light. This photograph is of various types of DNA that have been electrophoresed on the same gel. Note that high molecular weight DNAs do not separate well on this gel. This can be corrected by altering gel density.
Visualization and Quantitation Visualization: Staining: a- Ethidium bromide for DNA, then visualize bands or zones by a transilluminator. b-bromophenol blue or commasssie blue for proteins. c- Ninhydrin for amino acids. d- Sudan black for lipoproteins. e- Iodine for polysaccharides.
Quantitation Direct densitometry. OR Eluting the dye followed by spectrophotometric measurment
Densitometric representation of a protein electrophoresis gel
Limitations and errors in routine electrophoresis 1- Wick Flow: Heat production Evaporation of solvent from the electrophoresis support. Buffer will be drawn into the support from both directions affect the migration of the separating molecules. This is avioded by: - Tight closure of the lid of electrophoresis. - Introduction of a cooling system.
Electroendosmosis1 - + + + + + - + + + + - - + - - - + - + + + + - - + + + + + - + + - - - - + + + + + - + + + - - Electrophoresis fixed immobile mobile homogenous support charges ions ion cloud solution electokinetic or zeta potential
Electroendosmosis2 It is the movement of solvent and its solute relative to a fixed support. Certain electrophoretic media ( as cellulose acetate in contact with water adsorb OH- ions which become fixed to the support. An ion cloud of mostly positive charges is formed (see figure in previous slide) The potential that exits between the fixed ions and the associated ion cloud is called electokinetic or zeta potential
Electroendosmosis3 When a current is applied to such a system, charges attached to the support remain fixed, but the cloud of ions in the solution is free to move to the pole of opposite polarity. Because the ions in solution are highly hydrated, this results in movement of the solvent as well. This will interfere with the migration of molecules. Molecules that would normally move in theopposite direction to this flow may remain immobile or even swept backwards to the opposite pole.
Electroendosmosis4 Electoendosmosis can be minimized by using an inert supporting material as agarose or PAG.
TYPES OF ELECTROPHORESIS Zone electrophoresis Capillary electrophoresis. Pulsed field electrophoresis. Iso-electric Focusing. Two-dimensional PAGE. Blotting Techniques.
Zone electrophoresis It is migration of charged molecules as zones in porous supporting medium. Cellulose acetate Agarose gel Polyacrylamide gel Membrane electrophoresis electrophoresis. (CAE) (AGE) (PAGE)
Cellulose Acetate Membrane Electrophoresis (CAE) Cellulose + acetic anhydride cellulose acetate dry, opaque, and brittle. Advantages: -rapid procedure (20-60minutes) -ability to store the results in membrane for long periods. Disadvantages: -need presoaking with buffer. - Need clearing before densitometric scanning - Not inert electroendosmosis
Agarose gel electrophoresis(age) Agarose is sulphate free agar. Agar is a polysaccharide of sulfated galactose. Advantages: * Inert little electroendosmosis. *Amount of sample applied is very small (0.6-3 µl). *Short electrophoretic time (30-90 minutes). *Native clarity permitting excellent densitometic scanning.
Polyacrylamide gel electrophoresis (PAGE). PAG is formed from polymerization of acrylamide by heating in presence of small amouts of bisacrylamide ( the cross-linking agent). Polymerization is initiated by ammonium persulphate TEMED. TEMED catalyzes the decomposition persulphate(s 2 O 8 2- ) to give a free radical,leading to polymerization of the gel.
Polyacrylamide gel electrophoresis (PAGE) 2. S 2 O 8 2- +e-(temed) SO 4 2- +SO 4 free radical R R + M RM free radical acrylamide monomer RM + M RMM and so on Oxygen removes free radicals and so PAG are degassed and put between 2 tight glass plates.
Capillary Electrophoresis Involves electrophoresis of samples in a narrow tube ( 50µm internal diameter and 300 µm external diameter) of PAG. Advantage: They reduce problems resulting from heating defects. Because of the small diameter of the tubing, there is a large surface to volume ratio which gives enhanced heat dissipation.
Capillary Electrophoresis
In the first dimension, proteins are resolved in according to their isoelectric points(pis)using isoelectric focusing (IEF) In the second dimension, proteins are separated according to their approximate molecular weight using sodium dodecyl sulfate polyacrylamide-electrophoresis (SDS-PAGE)
Pulsed field electrophoresis DNA above 30-50 kb migrates with the same mobility regardless of size. This is seen in a gel as a single large diffuse band. If, however, the DNA is forced to change direction during electrophoresis, different sized fragments within this diffuse band begin to separate from each other. The smaller the molecule the faster it will realign itself.
Blotting Techniques. Southern blotting for DNA. Northern blotting for RNA. Western blotting for protein.
Southern blotting