Electrophoresis

Aneeqa Naseer 2011-ag-3886Ayesha Javed 2011-ag-3889Sadia Manzoor 2011-ag-3944

Contents:

1) Why electrophoresis??2) What is electrophoresis??3) Principle4) Agarose gel electrophoresis5) Sds-PAGE (Polyacrylamide gel electrophoresis)6) Factors effecting electrophoresis7) applications

Why electrophoresis?

To separate DNA fragments from each other

To determine the sizes of DNA fragments

To determine the presence or amount of DNA

To analyze restriction digestion products.

Determination of molecular weight of proteins.

What is electrophoresis? It describes migration of charged

particles or molecules under the influence of electric field.

It is standard method for separation, identification, analysis and purification of:– DNA molecules– protein molecules

PRINCIPLE

separates molecules from each other on the basis of– size – charge – shape

basis of separation depends on how the sample and gel are prepared?

Gel electrophoresis

What is a gel? Gel is a cross linked polymer whose

composition and porosity is chosen based on the specific weight and porosity of the target molecules.

Types of Gel: Agarose gel Polyacrylamide gel

Agarose gel electrophoresis

Agarose gel: Agarose is extracted in the form of agar from several species of red

marine algae, or seaweed, found in California and eastern Asia, dissolves in near-boiling water, and forms a gel when cools.

Has gelling temperature 35-38˚C and melts at 90-95 ˚C. Used to separate macromolecules such as nucleic acids, large

proteins and protein complexes.

Composition: It is prepared by dissolving 0.5% agarose in boiling water and

allowing it to cool to 40°C. It is fragile because of the formation of weak hydrogen bonds and

hydrophobic bonds.

Material required for gel electrophoresis

Electrophoresis chamber Agarose gel Gel casting tray Buffer Staining agent (dye) A comb DNA ladder Sample to be separate

Electrophoresis Equipment

Gel Casting Trays available in a variety of

sizes and composed of UV-transparent plastic.

The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis.

Applied voltage

• voltage, rate of migration• The higher the voltage, the more quickly the

gel runs• But if voltage is too high, gel melts• The best separation will apply voltage at no

more than 5V/cm of gel length.

Buffers During electrophoresis water undergoes

hydrolysis : H2O H + OH- Buffers prevent the pH from changing by

reacting with the H+ or OH- products Most common buffer used is called TRIS

– [tris (hydroxymethyl) aminomethane]

Buffers (cont.)

Another compound is added to make Tris an effective buffer — either boric or acetic acid

Another compound is added to bind metals EDTA

The buffer is either TBE or TAE

TBE is made with Tris/Boric Acid/EDTA

TAE is made with Tris/Acetic Acid/ EDTA

Staining of DNA To make DNA fragments visible after electrophoresis, the DNA must be

stained The favorite—ethidium bromide When bound to DNA it fluoresces under ultraviolet light (reddish –

orange color) Convenient because it can be added directly to the gel Sensitive—detects 0.1ug of DNA. ethidium bromide is mutagenic so care must be taken while handling

the dye. Other alternatives for ethidium bromide :

Methylene blue Syber safe xylene cyanol bromphenol blue

A Comb A comb is placed in the

liquid gel after it has been poured

Removing the comb from the hardened gel produces a series of wells used to load the DNA

DNA ladder It is a solution of DNA

molecules of different length DNA Ladder consists of known

DNA sizes used to determine the size of an unknown DNA sample.

The DNA ladder usually contains regularly spaced sized samples which when run on an agarose gel looks like a "ladder".

Method For Electrophoresis

Add running buffer, load samples and marker

Run gel at constant voltage until band separation occurs

Pour into casting tray with comb and allow to solidify

View DNA on UV light box and show results

Prepare agarose gelMelt, cool and add Ethidium Bromide. Mix thoroughly.

Get your sample obtained from previous purifying technique (i.e. PCR)

Set up gel, remove comb

Load Buffer

Load Sample

Run Gel

Stain and look at with UV light

DNA is negatively charged.

+-

Power

DNA

When placed in an electrical field, DNA will migrate toward the positive pole (anode).

H

O2

An agarose gel is used to slow the movement of DNA and separate by size.

SDS Since we are trying to separate many different

protein molecules of a variety of shapes and sizes, we first want to get them to be linear so that the proteins no longer have any secondary, tertiary or quaternary structure.

SDS (cont.)

To have proteins with linear structures we use sodium dodecyl sulfate (SDS).

SDS is a detergent that can dissolve hydrophobic molecules, also have negative charge (sulphate) attached to it allowing it to run properly through the gel (from negative to positive).

If cell is incubated with ito membranes will be dissolvedo Proteins will be solubilized by ito All proteins covered with negative charges

End result has two important features: 1. Proteins contain only primary structures.2. In electric field migrate towards positive pole.

Why PAGE?

Proteins separate only on the basis of charges in electric field.

We also need to separate it on basis of size. To separate it on basis of size we use PAGE.

It is polymer of acrylamide monomers when its polymer is formed it turns into gel and we use electricity to pull proteins through the gel, process is called polyacrylamide gel electrophoresis.

Polyacrylamide gel

Tunnels of different diameters

What is PAGE?

Polyacrylamide gel

Polyacrylamide is a polymer of acrylamide monomers.

Like Agarose Gels, Polyacrylamide gels are used to separate protein molecules by shape, size and charge.

Polyacrylamide is specifically used for proteins because it provides the protein with an environment where it will not become denatured.

Allowing different sized proteins to move at different rates.

Polyacrylamide gel When electricity passed proteins tend to move

through gel in bunches, or bands, since there are so many copies of each protein and they are all same size.

Controlled time is given for run so that proteins not reached to other side of the gel.

After it stain proteins. Bands

SDS-PAGE

Gel has 5 number lanes where 5 different samples of proteins are applies to gel.Lane 1: molecular weight standards of known sizes.Lane 2: mixture of 3 proteins of different sizes with a being biggest and c being smallest.Lane 3: protein a by itselfLane 4: protein b by itselfLane 5: protein c by itself

Factors affecting electrophoresis

• 1.The sample• 2. DNA or RNA Molecular Weight• 3. Voltage• 4. Agarose• 5. Buffer• 6. Visualisation

The Sample:

Charge/mass ratio of the sample dictates its electrophoretic mobility. The mass consists of not only the size (molecular weight) but also the shape of the molecule.

• Charge: The higher the charge, greater is the • electrophoretic mobility. The charge is dependent on pH of the

medium. b) Size: The bigger molecules have a small electrophoretic mobility compared to the smaller particles. c) Shape: The globular protein will migrate faster than the fibrous protein

DNA or RNA Molecular Weight The length of the DNA molecule is the most important factor, smaller molecules travel

farther. voltage The higher the voltage, the faster the DNA moves.

But voltage islimited by the fact that it heats and ultimately causes the gel to melt. High voltages also decrease the resolution (above about 5 to 8 V/cm)

Agarose

• Agarose gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pair to several megabases (millions of bases) using specialized apparatus. Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules.

• The distance between DNA bands of a given length is determined by the percent agarose in the gel. In general lower concentrations of agarose are better for larger molecules because the result in greater separation between bands that are close in size. The disadvantage of higher concentrations is the long run times (sometimes days). Instead high percentage agarose gels should be run with a pulsed field electrophoresis (PFE), or field inversion electrophoresis.

Buffer• The most common buffers for agarose gel: TAE: Tris acetate EDTA TBE: Tris/Borate/EDTA SB: Sodium borate. • TAE has the lowest buffering capacity but provides the best resolution for

larger DNA. This means a lower voltage and more time, but a better product.

visualizationEthidium bromide dye which is used as staining dye, in staining is carcinogen.

applications

• Vast applications includei. vaccine analysisii. DNA analysisiii. Protein analysisiv. Antibiotic analysis

Vaccine analysis

• Electrophoresis is widely used in vaccine analysis method.

• Several vaccines have been purified, processed and analysed.

e.g. influenza vaccine, polio vaccine, hepatitis vaccine.

DNA analysis

• Through electrophoresis specific DNA sequences can be analysed, isolated and cloned.

• Analysed DNA may be used in forensic investigations and paternity tests.

Protein analysis

• Electrophoresis has advanced our understanding on the structure and function of protein.

• Amount of protein in your urine and blood is measured and compared to established normal values- lower or higher than the normal levels usually indicates a disease.

Antibiotic analysis

• . With electrophoresis, experts are not only able to synthesize new antibiotics but are also able to analyze which types of bacteria are antibiotic-resistant.

• These drugs, such as penicillin, are among the widely prescribed drugs against bacterial infections