2 Purposes To understand the principle of Gel electrophoresis To become familiar with the part of the electrophoresis setup
3 What is Electrophoresis? Electrophoresis is a laboratory technique for separating molecules based on their charge.
4 Separation of a Mixture of Charged Molecules Charged molecules are separated based on their electrical charge and size.Charge SeparationSize SeparationAnalyzeIdentifyPurifyMixture of Charged MoleculesPositive MoleculesNegative Molecules
5 How Separation Occurs 1- Electrical Charge: Many molecules (amino acids, proteins, DNA, and RNA) have naturally occurring negative and positive charges on them.The sum of these charges determines the overall charge.Gel concentrationShapeVoltageInteraction with agaroseMolecules with a negative charge (anions) will be attracted to the positively charged node (anode).Molecules with a positive charge (cations) will be attracted to the negatively charged node (cathode).
6 Proteins Entering Porous Material How Separation Occurs2- Molecule Size:The porous material is made of microscopic particles suspended in a gel.The microscopic particles attach to one another forming tunnels that act as a sieve to separate the molecules.Small molecules can move faster than large molecules.Size to molecular ratioPorous MaterialProteins Entering Porous MaterialSmallest Move Fastest
7 Gel ElectrophoresisGels can be made from substances such as agarose or polyacrylamide.Agarose – a complex sugar chain from red seaweed.It is commonly used in foods (ice cream, and jellies) and many biological mediums.It has a large pore size good for separating large molecules quickly.Polyacrylamide – chain of acrylamide molecules.It is often used to make plastics and rubber.It has a small pore size good for separating small molecules.Red Sea WeedAcrylic Acid
8 Agarose Gel 1% agarose 2% agarose A porous material derived from red seaweedAgarose is highly purified to remove impurities and chargeActs as a sieve for separating molecules.This solid matrix will allow the separation of fragments by size.Concentration affects molecules migrationLow conc. = larger pores better resolution of larger DNA fragmentsHigh conc. = smaller pores better resolution of smaller DNA fragments1% agarose2% agarose
9 Fragment ResolutionGel Concentration – Is dependant upon the size of the DNA fragments to be separated.
10 + - Agarose at Room Temperature is a 3-Dimentional solid matrix. The smaller the fragments the further the migration or movement through the matrix.+-Powersmalllarge
11 Purposes for Agarose Gel Electrophoresis Analysis of molecules sizeSeparation and extraction of moleculesQuantification of molecules
13 Components of an Electrophoresis System Power supply and chamber, a source of power supplyBuffer, a fluid mixture of water and ionsAgarose gel, a porous material that molecules migrates throughGel casting materialsIons: atoms that have a positive or negative charge because they have lost or gained electrons.Electrophoresis: migration of ions at different speeds is a basic principal
14 - + Cathode Anode Buffer Power Supply Dyes During electrophoresis, water is electrolyzed which generates protons (H+ ions)at the anode (positive) and hydroxyl ions (OH -1)at the cathode (negative). The cathode (negative) end of the electrophoresis chamber then becomes basic and the anode (positive) end becomes acidic.The electrode at which electrons enter the gel box from the power supply (along the black wire) is called the cathode and is negative (-). The electrode at which electrons leave the box and re-enter the power supply (along the red wire) is called the anode and carries a positive charge (+). The flow of electrons sets up a potential energy difference between the electrodes. This is known as potential, and is measured in volts. It establishes an electric field through which the ions in the gel box fluid migrate. The migration of ions in the fluid creates electrical current which is measured in milliamperes (milliamps).Anode+BufferPower SupplyDyes
15 Electrophoresis Equipments Power supplyCoverGel tankElectrical leadsCasting trayGel combs
16 Electrophoresis Buffer TAE (Tris -acetate-EDTA) and TBE (Tris-borate- EDTA) – pH bufferTris Acetic acid provide ions to support conductivity and maintain pHEDTA, prevent brake down of moleculesConcentration affects DNA migrationUse of water will produce no migratonHigh buffer conc. could melt the agarose gelA buffer is a chemical system that maintains a relatively constant pH even when strong acids or bases are added. Buffer solutions contain either a weak acid or weak base and one of their salts. Because a change in pH can alter the charge on a particle, it is important to use a buffer solution when separating during electrophoresis.
17 Overview of Agarose Gel Electrophoresis Gel PreparationLoading the gelRunning the gel
18 Gel Preparation Agarose is a linear polymer extracted from seaweed.
19 Agarose Buffer Solution Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.
20 Melting the Agarose Agarose is insoluble at room temperature (left). The agarose solution is boiled until clear (right).Gently swirl the solution periodically when heating to allow all the grains of agarose to dissolve.***Be careful when boiling - the agarose solution may become superheated and may boil violently if it has been heated too long in a microwave oven.
23 Pouring the gelAllow the agarose solution to cool slightly (~60ºC) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubbles.
24 When cooled, the agarose polymerizes, forming a flexible gel When cooled, the agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the comb.