Electrophoresis

Electrophoresis

• Separation technique based on the movement of analyte through a conductive medium in response to an applied electrical field.• The medium is usually a buffered aqueoussolution.• In the absence of other factors, cationic species will migrate towards the cathode and anionic species towards the anode.

Theory of Electrophoresis

Dr Gihan Gawish3

Electrophoretic separations are based upon the fact that the electrical force

(F) on a charged particle (ion) in an electrical field (E) is proportional to

the charge of the particle (q),

F = qEThe migration of the charged particle in the electric field, called the

electrophoretic mobility (μ),

μ = v/E = q/f

Theory of Electrophoresis

4

Move at quite different depending on

physical characteristics of the molecule i.e its molecular size.

TYPES OF ELECTROPHORESISFree-Solution method/ capillary eclectrophoresis.

! Absence of a supporting/stabilizing medium.! Sample is introduced into a tube filled with abuffering liquid.! A field is applied and species migrate based ontheir charge to mass ratios.! Tiselius - 1948 Nobel prize for his development of this approach for the purification of proteins.

Most popular method is capillary electrophoresis

1- Moving Boundary Electrophoresis

Electrophoresis in a free solution.

the separation of colloids through electrophoresis

the motion of charged particles through a stationary liquid under the influence of an electric field.

Developed by Arne Tiselius in 1937. Tiselius was

awarded the 1948 Nobel Prize in chemistry for his

work

Stabilizing media methods•Presence of a supporting medium like paper,•packing, or gel.

•Resemble chromatographic methods except that•migration is based on an electrical field instead of•a mobile phase.

•A number of methods have been based on this approach including

•Electrochromatography•Zone electrophoresis•Electromigration•Ionophoresis

2- Zone Electrophoresis

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A drop of sample is applied in a band to a thin sheet of supporting material, like paper, that has been soaked in a slightly-alkaline salt solution.  

Paper electrophoresis employs filter paper strips soaked in buffer solution, usually diethylbarbituric acid and barbituric acid dissolved in alkali (Veronal buffer), pH 8.6. A small volume of serum is placed on the paper and a direct current passed for several hours

3- Gel Electrophoresis

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The term "gel" in this instance refers to the matrix used to contain, then separate the target molecules.

In most cases the gel is a cross linked polymer whose composition and porosity is chosen based on: the specific weight composition of the target to be

analyzed.

Introduction Principles of nucleic acid separation by agarose gel electrophoresisAgarose gel electrophoresis is a routinely used method for separating proteins, DNA or RNA. Nucleic acid molecules are size separated by the aid of an electric field where negatively charged molecules migrate toward anode (positive) pole. The migration flow is determined solely by the molecular weight where small weight molecules migrate faster than larger ones. In addition to size separation, nucleic acid fractionation using agarose gel electrophoresis can be an initial step for further purification of a band of interest. Extension of the technique includes excising the desired “band” from a stained gel viewed with a UV transilluminator

Gel Electrophoresis

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*Acrylamide, in contrast to *Polyacrylamide, is a

neurotoxin and must be handled using

appropriate safety precautions to avoid

poisoning.

larger nucleic acids (greater

than a few hundred bases)

the preferred

matrix *agarose.

VisualizationEthidium bromide is the common dye for nucleic acid visualization. The early protocol that describes the usage of Ethidium bromide (2,7-diamino-10-ethyl-9- phenylphenanthridiniumbromide-) for staining DNA and RNA in agarose gels dates as far back as 1970s.

Although with a lower efficiency compare to the double- stranded DNA, EtBr is also used to stain single- stranded DNA or RNA.

Under UV illumination, the maximum excitation and fluorescence emission of EtBr can be obtained from 500- 590 nm. Exposing DNA to UV fluorescence should be performed rapidly because nucleic acids degrade by long exposures and thus, the sharpness of the bands would be negatively affected.

Gel Electrophoresis- Visualization

Dr Gihan Gawish13

stained

Ethedium bromide Silver

coomassie blue

UVRadioacti

vity

Photograph Autoradiogram

The bands observed (unknown molecular weight) can be compared to those of the known (Molecular weight size markers) in order to determine their size.

Molecular weight size markers contain a mixture of molecules of known sizes.

marker run on one lane in the gel parallel to the unknown samples

Gel Electrophoresis- Visualization

Bands in different

lanes that end up at the

same distance

from the top

contain molecules that passed

through the gel with the same

speed

means they are approximately the same size

An alternative dsDNA stain is SYBR Green I. Despite the fact that SYBR Green is more expensive, it is 25 times more sensitive than ethidium bromide

Since EtBr stained DNA is not visible in natural light, negatively charged loading buffers are commonly added to DNA prior to loading to the gel.

Loading buffers are particularly useful because they are visible in natural light and they co-sediment with DNA.

Xylene cyanol and Bromophenol blue are the two common dyes used as loading buffers and they run about the same speed as DNA fragments that are 5000 bp and 300 bp respectively. The other less frequently used progress markers are Cresol Red and Orange G which run at about 125 bp and 50 bp, respectively

Agarose GelStained with ethidium bromide (EtBR) to Visualize the DNAAgarose GelStained with ethidium bromide (EtBR) to Visualize the DNA

Screening PCR products to test for the presence of specific DNA sequences

500 bp

molecularweight

markers

molecularweight

markers

correctPCR

product

600 bp700 bp

1000 bp

slots whereDNA is loaded

17 Dr.Saba Abdi

Preparing and running standard agarose DNA gels

Several electrophoresis buffers can be used for fractionating nucleic acid such as, Trisacetate- EDTA (TAE) Tris-borate-EDTA (TBE)

For gel preparation, Agarose powder electrophoresis grade is mixed with electrophoresis buffer to the desired concentrations (usually with a range of 0,5-2%) then heated in a microwave oven until completely dissolved. Ethidium bromide is usually added to the gel for nucleic acid visualization. The mixture is cooled to 60 C and poured into the casting tray for solidification.

Wells should be placed towards the negative electrode?

At the same time, ethidium bromide migrates in the reverse direction, meets and couples with DNA fragments. DNA fragments are visualized by staining with ethidium bromide when adequate migration has occurred. Then, this fluorescent dye intercalates between bases of DNA and RNA

Factors listed below are effecting the mobility of DNA fragments in agarose gels

Agarose concentration

In the gel, the distance between DNA bands of a given length is determined by the percent agarose. Higher concentrations have the disadvantage of long run times.

Most agarose gels are prepared with the agarose concentrations ranging 0.7% (good separation or resolution of large 5–10kb DNA fragments) to 2% (good resolution for small0.2–1kb fragments)

VoltageMigration of fragments in an agarose gel depends on the difference in electric current.Different optimal voltages are required for different fragment sizes. For instance, the best resolution for fragments larger than 2 kb could be obtained by applying no more than 5 volts per cm to the gel

Electrophoresis bufferVarious buffers are used for agarose electrophoresis. The two most common buffers for nucleic acids are Tris/Acetate/EDTA (TAE) and Tris/Borate/EDTA (TBE). DNA fragments migrate with different rates in these two buffers due to differences in ionic strength. Buffers not only establish an ideal pH, but provide ions to support conductivity. In general, the ideal buffer should produce less heat, have a long life and a good conductivity.

For example, deviations from the optimal concentration of the buffer (over concentrated) could produce enough heat to melt the gel

Applications of Gel Electrophoresis

The results can be

analyzed quantitatively by

visualizing the gel with UV

light and a gel imaging

device.

The image is recorded with

a computer operated

camera

Forensics,

Molecular

biology,

Genetics

Microbiology

Biochemistry.