AGAROSE GEL ELECTROPHORESIS
Objectives:- To understand the principle of extraction and gel electrophoresis.
Principle:-
> The term electrophoresis describes the migration of a charged particle under the influence of an electric field. Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations (+) or anions (-). Under the influence of an electric field these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.
> The equipment required for electrophoresis consists basically of two items, a power pack and an electrophoresis unit. Electrophoresis units are available for running either vertical or horizontal gel systems. Vertical slab gel units are commercially available and routinely used to separate proteins in acrylamide gels. The gel is formed between two glass plates that are clamped together but held apart by plastic spacers.
> Agarose is a linear polysaccharide (average relative molecular mass about 12 000) made up of the basic repeat unit agarobiose, which comprises alternating units of galactose and 3,6- anhydrogalactose. Agarose is one of the components of agar that is a mixture of polysaccharides isolated from certain seaweeds. Agarose is usually used at concentrations of between 1% and 3%.
> Agarose gels are formed by suspending dry agarose in aqueous buffer, then boiling the mixture until a
clear solution forms. This is poured and allowed to cool to room temperature to form a rigid gel. The
gelling properties are attributed to both inter- and intramolecular hydrogen bonding within and between
the long agarose chains. This cross-linked structure gives the gel good anticonvectional properties. The
pore size in the gel is controlled by the initial concentration of agarose; large pore sizes are formed
from low concentrations and smaller pore sizes are formed from the higher concentrations.
> Electrophoresis in acrylamide gels is frequently referred to as PAGE, being an abbreviation for
polyacrylamide gel electrophoresis. Cross-linked polyacrylamide gels are formed from the
polymerisation of acrylamide monomer in the presence of smaller amounts of N,N’-methylenebisacrylamide (normally referred to as ‘bis’-acrylamide). Note that bisacrylamide is essentially two acrylamide molecules linked by a methylene group, and is used as a cross-linking agent. > Acrylamide monomer is polymerised in a head-to-tail fashion into long chains and occasionally a bis-acrylamide molecule is built into the growing chain, thus introducing a second site for chain extension. Proceeding in this way a cross-linked matrix of well-defined structure is formed. The polymerisation of acrylamide is an example of free-radical catalysis, and is initiated by the addition of ammonium persulphate and the base N,N,N’,N’-tetramethylenediamine (TEMED).
Materials & Reagents:-
.bmp)
Procedure:-
1. Load dye samples into the wells (also called lanes) from left to right following the order listed below.
To load the first sample (bromphenol blue) into the well, draw 20 μl of dye.
Order of Loading:-
lane 1 bromphenol blue
lane 2 methyl orange
lane 3 ponceau G
lane 4 xylene cyanol
lane 5 pyronin Y
lane 6 unknown #1
lane 7 unknown #2
lane 8 unknown #3
2. Once all the dye samples have been loaded, place the lid on the electrophoresis chamber. Orient the lid with the positive end of the chamber connected to the red (positive) cord and the negative end of the chamber connected to the black (negative) cord. Then connect the electrical cords to the power supply, with the positive lead in the positive input (red to red) and the negative lead in the negative input (black to black). If using a multi-channelled power supply, make sure both electrical leads are connected to the same channel.
3. Turn on the power supply and set it to the desired voltage. Watch as the dyes slowly move into the gel and separate over time. Do not allow any of the dyes to run off the gel. Run the gel until the band in lane 3 is 0.5 cm from the end of the gel.
4. Once the desired separation of dyes has been achieved, turn off the power, disconnect the leads from the inputs, and remove the top of the electrophoresis chamber.
5. Carefully remove the casting tray and slide the gel into the plastic tray.