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: Electrical filed. 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. DNA is negatively charged

Separation of a Mixture of Charged Molecules Charged molecules are separated based on their electrical charge and size. Charge Separation Size Separation Analyze Identify Purify Mixture 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.

Proteins Entering Porous Material Molecule Size Porous Material Proteins Entering Porous Material Smallest Move Fastest

Components of an Electrophoresis System Power supply and chamber: a source of power supply Agarose gel: a porous material that molecules migrates through Buffer: a fluid mixture of water and ions. Gel casting materials Power Supply

- + Cathode Anode Buffer 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 + Buffer Dyes

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 "Most agarose gels are made with between 0.7% (good separation or resolution of large 5–10kb DNA fragments) and 2% (good resolution for small 0.2–1kb fragments) agarose dissolved in electrophoresis buffer. Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case. Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set evenly. 1% gels are common for many applications. Agarose gels do not have a uniform pore size, but are optimal for electrophoresis of proteins that are larger than 200 kDa.

Electrophoresis Buffer TAE (Tris -acetate-EDTA) and TBE (Tris-borate-EDTA) – pH buffer. Tris a pH buffer. Acetic acid provide ions to support conductivity and maintain pH. 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 A 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.

Overview of Agarose Gel Electrophoresis Gel Preparation Loading the gel Running the gel

Gel Preparation Agarose is a linear polymer extracted from seaweed.

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

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.

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

Principle 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: all proteins contain only primary structure and 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

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 ammonium persulphate and the base N,N,N’,N’-tetrametyhlenediamine (TEMED)

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 Stain with Silver stain