ELECTROPHORESIS

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Principles:

Electrophoresis is the process of moving charged molecules in solution by applying an electric field across the mixture.

Basics

Speed dependent on their charge, shape, and size, hence electrophoresis has been extensively developed for molecular separations. It is used chiefly for analysis and purification of very large molecules such as proteins and nucleic acids,

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Factors Affecting Electrophoresis

1.

The Electric Field.

1. Voltage. b. Current. c. Resistance.

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Factors Affecting Electrophoresis

2. The Sample a. Charge. b. Shape.

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Factors Affecting Electrophoresis

3. The Buffer a. Composition. b. Concentration. c. pH.
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Factors Affecting Electrophoresis

4. The Supporting Medium. a. Adsorption. b. Electro osmosis. c. Molecular Sieving.
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Gel Electrophoresis.

Separation of high molecular weight substances such as proteins and nucleic acids Improved resolution since the gel is water insoluble and hydrophilic.

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Gel Electrophoresis.

Eg: Starch, agar and polyacrylamide. Molecular sieving property of gels helps to separate large ionic compounds having similar charge but different size and shape.

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Starch Gel Electrophoresis

Starch gels are prepared by heating and cooling mixture of partially hydrolysed starch resulting in a semi rigid gel formation. Good for separating complex mixtures of structural molecules and physiologically active proteins. Analysis of isoenzyme patterns (zymograms)

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Agarose Gel Electrophoresis

Agar is a cheap, non toxic, chemically ill defined, complex, powdery mixture. Contains two galactose based polymers, agarose and agaropectin.

Agarose Gel Electrophoresis

Dissolves in boiling aqueous buffers and forms a gel at 38C. Large pore size and low frictional resistance. Facilitates separation of macromolecules. Suitable for chemical staining after separation. Excellent for detection of antigenic proteins by isotachophoresis and immunoelectrophoresis.

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DNA separation using Agarose Gel Electrophoresis

Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Agarose gel electrophoresis is routinely used for the preparation and analysisto edit Master subtitle style Click of DNA.

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Equipment
1.

A power pack. Supplies direct current between the electrodes between the electrophoresis unit.

Equipment
2.

Electrophoresis Unit Electrodes. Buffer reservoirs. Support medium. Transparent insulating cover

a. b. c. d.

 DNA is negatively charged. When placed in an electrical field, DNA will migrate toward the positive pole (anode). An agarose gel is used to slow the movement of DNA and separate by size.

O 2

DNA

Powe r
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Polymerized agarose is porous, Scanning Electron allowing for theAgarose Gel Micrograph of

How fast will the DNA migrate? of the electrical field, buffer, density of agarose strength
gelof the DNA! Size *Small DNA move faster than large DNA gel electrophoresis separates DNA according to size

DN A small large

Powe r
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Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

Making an Agarose Gel

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Agaros e
Dgalactose 3,6-anhydro L-galactose

Sweetened agarose gels have been eaten in the Far East since the 17th century.

Agarose is a linear polymer extracted from seaweed.

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*Lina Hesse, technician and illustrator for a colleague of Koch was the first to suggest agar for use in culturing bacteria

Agarose was first used in biology when Robert Koch* used it as a culture medium for Tuberculosis bacteria in 1882

An agarose gel is prepared by combining agarose powder and a buffer solution.

Buffer

Flask for boiling

Agarose
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Electrophoresis Equipment
Power supply Gel tank Cover Electrical leads

Casting tray Gel combs

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Gel casting tray & combs

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Preparing the Casting Tray

Seal the edges of the casting tray and put in the combs. Place the casting tray on a level surface. None of the gel combs should be touching the surface of the casting tray. 7/3/12

Agaros e

Buffer Solution

Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.
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Melting the Agarose

Gently swirl the solution periodically when heating to allow all the grains of agarose to dissolve. ***Be careful when boiling - the agarose solution may 7/3/12 become superheated and may boil violently if it has

Agarose is insoluble at room temperature (left). The agarose solution is boiled until clear (right).

Pouring the gel

Allow the agarose solution to cool slightly (~60C) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubbles.
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Each of the gel combs should be submerged in the melted agarose solution.
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When cooled, the agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the combs and tape. 7/3/12

Place the gel in the electrophoresis chamber.

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DNA buffer Anode (positive )

well s

Cathod e (negative)

Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.
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Sample Preparation
Mix the samples of DNA with the 6X sample loading buffer (w/ tracking dye). This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells.

6X Loading Buffer: Bromophenol Blue (for color) Glycerol (for weight)

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Loading the Gel

Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.
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Running the Gel

Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.
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Cathod e (-)
DN A (-)

wells Bromophenol Blue

Gel Anod e (+)

After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.
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DNA Ladder Standard

12,000 bp 5,000

2,000 Note: 1,650 bromophenol 1,000 blue migrates 850 at 650 approximately 500 bromophenol 400 the same rate blue 300 as a 300 bp + 200 DNA molecule DNA ladder (DNAs of know sizes) on the 100 Inclusion of a gel makes it easy to determine the sizes of unknown 7/3/12 DNAs.

DNA migratio n

As an alternative to purchasing costly DNA ladders, one can be created using meal worm (Tenebrio molitor) DNA and a restriction enzyme.

7/3/12 http://people.uis.edu/rmosh1/DNAexerciseVIIa02.pdf

Staining the Gel Ethidium bromide binds to DNA and fluoresces under UV
light, allowing the visualization of DNA on a Gel. Ethidium bromide can be added to the gel and/or running buffer before the gel is run or the gel can be stained after it has run.

***CAUTION! Ethidium bromide is a powerful mutagen and 7/3/12 is moderately toxic. Gloves should be worn at

Safer alternatives to Ethidium Bromide Methylene Blue BioRAD - Bio-Safe DNA Stain

Wards - QUIKView DNA Stain

Carolina BLU Stain
advantages

others Inexpensive

disadvantages

Less toxic No UV light required No hazardous waste disposal

Less sensitive More DNA needed on gel Longer staining/destaining time

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Staining the Gel

Place the gel in the staining tray containing warm diluted stain. Allow the gel to stain for 25-30 minutes. To remove excess stain, allow the gel to destain in water. Replace water several times for efficient destain. 7/3/12

Ethidium Bromide requires an ultraviolet light source to visualize

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Visualizing the DNA (ethidium bromide)

DNA ladder 1

DNA ladder 7 8

wells 5,000 bp 2,000 1,650 1,000 850 650 500 400 300 200 100

PCR Product Primer dimers

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Samples # 1, 4, 6 & 7 were positive for Wolbachia DNA

Visualizing the DNA (QuikVIEW stain)

DNA ladder

wells PCR Produ ct

+ - - - - + + - - + +

2,000 bp 1,500 1,000 750 250 500

Samples # 1, 6, 7, 10 & 12 were positive for Wolbachia 7/3/12 DNA March 12, 200

Polyacrylamide Gel Electrophoresis.

Polyacrylamide gels are physically tougher than agarose gels. The gel forms when a mixed solution of acrylamide and cross-linker monomers co-polymerize into long chains that are covalently cross-linked. The most common cross-linker is N,N'methylenebisacrylamide (bis).

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Polyacrylamide Gel Electrophoresis.

Porosity of gel is determined by relative proportion of acrylamide monomer to cross linking agent Gels may be defined in terms of total % of acrylamide present.

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SDS PAGE

Sodium dodecylsulphate polyacrylamide gel electrophoresis. Principle: SDS is an anionic detergent which binds to proteins causing their denaturation. In excess of SDS the proteins have a constant negative charge.

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SDS PAGE

Protein-SDS complex moves towards anode. Mobility inversely proportional to log of molecular weight. Molecular weights of unknown proteins can be determined.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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Polyacrylamide Gel Electrophoresis.

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