Electrophoresis

Electrophoresis

Principles of Gel Electrophoresis

Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used techniques in biochemistry and molecular biology.
When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode.
Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most commonly, the gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value.
The gel itself is composed of either agarose or polyacrylamide, each of which have attributes suitable to particular tasks:

Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are extremely easy to prepare: you simply mix agarose powder with buffer solution, melt it by heating, and pour the gel. It is also non-toxic.

Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques.
Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%.

Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymerization process, they must be poured between glass plates (or cylinders).
Acrylamide is a potent neurotoxin and should be handled with care! Wear disposable gloves when handling solutions of acrylamide, and a mask when weighing out powder. Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be handled with gloves due to the possible presence of free acrylamide.
Polyacrylamide gels have a rather small range of separation, but very high resolving power. In the case of DNA, polyacrylamide is used for separating fragments of less than about 500 bp. However, under appropriate conditions, fragments of DNA differing is length by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins.


Cross section diagram of gel apparatus design:
A. Tube
B. Vertical Slab
C. Horizontal submarine
D. Horizontal thin layer with buffer pads



Principle of Separation
• According to charge
• According to size

(1) According to charge
• When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge.
Instruments and Reagents
• Buffer
• Power supply
• Supporting media
• Detection and Quantification

I. Buffer
• Function of buffer
• 1. carries the applied current
• 2. established the pH
• 3. determine the electric charge on the solute
• High ionic strength of buffer
• produce sharper band
• produce more heat
• Commonly used buffer
• Barbital buffer & Tris-EDTA for protein
• Tris-acetate-EDTA & Tris-borate-EDTA (50mmol/L; pH 7.5-7.8)
II. Power Supply
• Function: To supply electric power
• Constant voltage (increasing of heat over time
• Heat = E x I x t
• Constant current
• according to Ohm’s law E= I x R
• R decrease over time, which inturn decrease the heat effect
E= EMF in volts (V)
I = Current in amperes (A)
t = Time in second (s)



III.Support materials used in electrophoresis
• Paper
• Starch
• Agar/agarose
• Cellulose acetate
• polyacrylamide gel
Support Media (ii)
• Agarose and polyacrylamide gels are across-linked, spongelike structure
• It is important that the support media is electrically neutral. Presence of charge group may cause:
• -Migration retardation
-The flow of water toward one or the other electrode so called ‘Electroendosmosis (EEO)’, which decrease resolution of the separation
(1) Agarose
• What is agarose?
• a chain of sugar molecules
• Extracted from seaweed
(1) Agarose gels (ii)
• For the separation of (1) large protein or protein complex (2) polynucleotide 50-30,000 base-pairs
The pore size is determined by adjusting the concentration of agarose in a gel (normally in the rank of 0.4-4% OH, O, CH2OH,

(2) Polyacrylamide gels
CH2=CHCONH2 + CH2(NHCOHC=CH2)2
Acrylamide N,N,N,N-methylenebisacrylamide
Free radical catalyst
-CH2-CH-CH2-CH-CH2-CH-
-CH2-CH-CH2-CH-CH2-CH-
CO, NH, CH2
A. Agarose gel B. Polyacrylamide gel
Polymerization of acrylamide gel can be initiated either by a chemical peroxide or by photochemical method
1. The most common method, ammonium persulphate as the initiator peroxide and TEMED as the catalyst
2. For photochemical polymerization, riboflavin and long-wave UV light are initiator and TEMED as the catalyst