Gel electrophoresis

Purposes• To understand the principle of Gel electrophoresis.

• To become familiar with the part of the electrophoresis setup.

• Gel electrophoresis: is a method used in

• Clinical chemistry to separate proteins by charge and or size.

• Biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length and to estimate the size of  DNA and RNA fragments.

• Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix.

• Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel.

• This phenomenon is called sieving.

Applications of Agarose Gel Electrophoresis

• Separation of restriction enzyme digested DNA including genomic DNA, prior to Southern Blot transfer. It is also often used for separating RNA prior to Northern transfer.

• Analysis of PCR products after polymerase chain reaction to assess for target DNA amplification.

• Allows for the estimation of the size of DNA molecules using a DNA marker or ladder which contains DNA fragments of various known sizes.

• Allows the rough estimation of DNA quantity and quality.

There are two components of electrophoresis system:

1. Electrical filed.2. The physical resistance of the matrix.

Electrical filed

• The sum of these charges determines the overall charge.• At neutral pH:

proteins have a unique electrical charged. both DNA and RNA bases are negative charged.

• Molecules with a negative charge (anions) will be attracted to the positively charged node (anode)……. Red color.

• Molecules with a positive charge (cations) will be attracted to the negatively charged node (cathode)…… Black color.

Charged molecules are separated based on their electrical charge and size.

Separation of a Mixture of Charged Molecules

Charge Separation

Size Separation

Analyze

Identify

PurifyMixture of Charged Molecules

Positive Molecules

Negative Molecules

The physical resistance of the matrix

• Electrophoresis is conducted through a gel substances called a matrix.

• Matrix acts as a physical barrier to movement of the substances.

• Without any resistance to there movement, “equally charged molecules will migrate through the electrical field at the same rate.

Molecule Size

Porous Material

Proteins Entering Porous Material

Smallest Move Fastest

Components of an Electrophoresis System

1. Power supply and chamber: a source of power supply

2. Agarose gel: a porous material that molecules migrates through

3. Buffer: a fluid mixture of water and ions.

4. Gel casting materials

Power Supply

Buffer

Dyes

+

-Cathode

Anode

Gel Electrophoresis

• They are many types of matrices used in electrophoresis, but all function similarly as physical barrier to movement.

Gels can be made from substances such as agarose or polyacrylamide.

• Agarose “ a complex sugar chain from red seaweed”.

– Non toxic carbohydrate.

– 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.

Agarose Gel• Agarose is highly purified to remove impurities and charge.

• Acts as a sieve for separating molecules.

• This solid matrix will allow the separation of fragments by size.

• Concentration affects molecules migration :-

• Low conc. = larger pores better resolution of larger DNA fragments

• High conc. = smaller pores better resolution of smaller DNA fragments

1% agarose

2% agarose

Electrophoresis Buffer

• TAE (Tris -acetate-EDTA) and TBE (Tris-borate-EDTA) – pH buffer.

1. Tris a pH buffer.

2. Acetic acid provide ions to support conductivity and maintain pH.

3. EDTA, prevent brake down of molecules. “all dissolved in water”.

Concentration affects DNA migration

• Use of water will produce no migraton

• High buffer conc. could melt the agarose gel

• Gel Preparation

• Loading the gel

• Running the gel

Overview of Agarose Gel Electrophoresis

Agarose is a linear polymer extracted from seaweed.

Gel Preparation

Agarose Buffer Solution

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

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.

Melting the Agarose

Loading the gel

• Gels are covered with a buffer solution. Prior to loading the samples.

• The DNA must be mixed with a loading dye. • The loading dye serves two purposes:

1. Increases the density of the DNA so it will sink into the wells.

2. Provides a visual marker so you know how far the DNA (which is not visible) has traveled in the gel.

Running the gel

Visualization

• DNA may be visualized using ethidium bromide which, when intercalated into DNA, fluoresce under ultraviolet light.

• While protein may be visualised using silver stain or Coomassie Brilliant Blue dye.

What is Wrong with Agarose Gels?

• Poor precision• Low sensitivity• Low resolution• Non-automated• Results are not expressed as numbers• Ethidium bromide staining is not very quantitative

• http://learn.genetics.utah.edu/content/labs/gel/

• http://www.youtube.com/watch?v=2UQIoYhOowM

• http://www.youtube.com/watch?v=ed-tQ7gAdnU

• http://highered.mcgraw-hill.com/sites/9834092339/student_view0/chapter20/electrophoresis.html

• http://www.dnalc.org/resources/animations/gelelectrophoresis.html

• http://www.youtube.com/watch?v=Fi63VjfhsfI• http://www.youtube.com/watch?v=QnbEQ8Au06U

Sodium DodecylSulphate- PolyAcrylamide Gel Electrophoresis

(SDS-PAGE)

• Polyacrylamide is a cross-linked polymer of acrylamide.

• Have smaller pores than agarose, therefore high degree of resolving power.

• Can separate DNA fragments which range in size from 10-500 bp.

• DNA fragments which differ in size by one nucleotide can be separated from each other.

Protein Electrophoresis

• Proteins, unlike DNA, do not have a constant size to charge ratio– In an electric field, some will move to the positive

and some to the negative pole, and some will not move because they are neutral.

• The purpose of this method is:• Separate proteins according to their size, and no other

physical feature. In order to understand how this works, we have to understand the two halves of the name: SDS and PAGE

• We need a way to convert all proteins to the same shape - we use SDS.

• SDS (sodium dodecyl sulfate) is a detergent that can dissolve hydrophobic molecules but also has a negative charge (sulfate) attached to it.

• The end result has two important features: 1. all proteins contain only primary structure and 2. all proteins have a large negative charge which means they

will all migrate towards the positive pole when placed in an electric field.

• They migrate through a gel towards the positive pole at a rate proportional to their linear size

Principle

Now we are ready to focus on the second half - PAGE

• PAGE is the preferred method for separation of proteins.

• Polymerization of acrylamide is initiated by the addition of TEMED.

• Bis-Acrylamide polymerizes along with acrylamide forming cross-links between acrylamide chains

Movement of Proteins in Gel

• smaller proteins will move through the gel faster while larger proteins move at a slower pace. bands.

• These can be transferred onto a nitrocellulose to be probed with antibodies and corresponding markers, such as in a western blot.

Vertical Gel Format: Polyacrylamide Gel Electrophoresis

Step by Step Instructions on how to assemble the polyacrylamide gel apparatus

Sample Buffer

• Tris buffer to provide appropriate pH• SDS (sodium dodecyl sulphate) detergent to dissolve

proteins and give them a negative charge• Glycerol to make samples sink into wells• Bromophenol Blue dye to visualize samples

Loading Samples & Running the gel