ElectrophoresisElectrophoresis

• Electrophoresis is one of the most important method for separating colloidal particles and biological molecules such as -

• Proteins • Carbohydrates • Nucleic acid• Polysaccharides• Peptides• Amino acids• Oligosaccharides• Nucleosides• Organic acids• Small anions and cat ions of body fluids .

Principle of Electrophoresis Principle of Electrophoresis

•Electrophoresis is a separation technique.•Use to separate out ionizable substances in a sample.• Based on the principle that –• A charged particle in solution will

migrate towards one of the electrodes when placed in an electrical field .

-Cations, positively charged particles will migrate to cathode.

-Anions , negatively charged particles will migrate to anode.

-Cations, positively charged particles will migrate to cathode.

-Anions , negatively charged particles will migrate to anode.

• The proteins found in plasma (TSP) all have amino acids as their subunits.

• So ,each protein has its own specific isoelectric point.

• Because of their different isoelectric points, each protein will move at a different rate when placed in an electrical field.

• Proteins with similar isoelectric points will migrate to a similar area in an electrical field

Serum protein electrophoresisHydragel – agarose gel

Serum protein electrophoresisHydragel – agarose gel

Serum proteins are separated into 6 groups: Albuminα1 - globulinsα2 - globulinsβ1 - globulinsβ2 - globulinsγ - globulins

Electrophoresis Electrophoresis

Process of separationProcess of separation

• When a voltage is applied across the electrodes, it generates a potential gradient.

• This potential gradient apply a force on charge bearing molecules.

• And drive a charged molecule towards an electrode.

• A frictional resistance oppose this movement of charged molecules .

• which depends on size, shape of molecules, pore size of medium and viscosity of the buffer.

• so, when a voltage is applied , molecules with different overall charges will begin to separate if they have different molecular sizes , shape and weight because experience different frictional forces.

Factors affect the rate of migration

Factors affect the rate of migration

• The mobility or rate of migration of ions in electrophoresis depends upon-

• Net charge of the molecule• Size and shape of the molecule• Support medium properties• Strength of the electrical field• Ionic strength of the buffer• Temperature

Separation of sample Separation of sample

1. According to charge

2. According to size

3. According to shape

According to chargeAccording to charge

1. When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge.

2 According to size • Smaller molecules run

faster through the medium than larger ones.

3. According to shape• Round shape molecules

have less frictional and electrostatic retardation than other shape molecules.

• so, moves fast in comparison to other shape molecules.

The Basic Components of electrophoresisThe Basic Components of electrophoresis

1. Electrophoresis chamber - is where it all process takes place. a. vertical b. horizontral

2. Electric power supply- provides the electricity that carries the

molecules through the gel.

3. Buffer solution - Buffer is chosen in that way

that effective separation take place.

4. Support medium – supporting medium is selected to ensure

accurate and effective separation.

Electrophoresis unitElectrophoresis unit

Electrophoresis unit, available for running either –

Electrophoresis unit, available for running either –

Vertical

Horizontal gel electrophoresis unit

Horizontal gel electrophoresis unit

Type of electrophoresisType of electrophoresis

• Vertical electrophoresis-• used for the seperation of proteins

in polyacrylamide gel

• Horizontal electrophoresis –• used for immunoelectrophoreis,

isolectric focusing, and for DNA , RNA in agarose gel.

1. Power supply

• The power supply is a source of constant voltage or current that provides energy to the electrodes.

• This drives the movement of the ions in the medium and results in the movement and separation of the molecules or solutes in the specimen.

2. Buffers2. Buffers

• The two important purposes of the buffer are - • a. To create the pH • b. And to conduct the current.

• Main features of buffer are-

• Buffer maintain a constant state of ionization of the molecules being separated.

• The pH set by the buffer determines the net charge on the solutes.

• the resulting net charge determines which electrode the solutes migrate toward.

• The buffer ions carry the current during electrophoresis.

• Besides setting the pH, the buffer also maintains the pH throughout the electrophoresis of the sample and control the conductivity of the gel.

• In pH 8 - 9, proteins will take on a negative charge and migrate to the anode.

• Most protein electrophoresis is performed at pH 8.6.

• Tris buffer (hydroxy methyl amino methane)- is a popular buffer for protein electrophoresis . It fix the pH at 8.6

• TBE buffer (Tris, Borate and EDTA) is often used for DNA fragments.

• To prevent microbial growth and contamination, buffers must be refrigerated when not in use.

• Using a buffer at refrigerator temperature also improves band resolution and lessens evaporation during electrophoresis.

3. Support Medium-• For electrophoretic separation of solutes,

the sample is placed on a support medium–

• It may be a- paper in paper electrophoresis and gel in gel electrophoresis

• which is in contact with buffer for separation.

kinds of support medium commonly used are :

kinds of support medium commonly used are :

• Starch

• Agar/agarose

• Cellulose acetate

• polyacrylamide gel (PAGE)

• Paper (cellulose)

Properties of gel -Properties of gel -• The gel is a polymer whose composition

and porosity is chosen based on the specific weight and composition of the sample to be analyzed.

• Agarose , and polyacrylamide gels are cross-linked, sponge like structure.

• It is important that the support media is electrically neutral.

•

• Cellulose acetate gel –

• Cellulose is chemically reacted with acetic anyhdride to form a cellulose acetate gel.

• Agarose gel is more often used than cellulose acetate gel for clinical electrophoresis.

• The gel pores allow for separation of proteins based on their charge and mass.

Agarose gel Agarose gel • A chain of sugar molecules• Extracted from seaweed

Agarose gel Agarose gel • For the separation of (1) large protein or protein complex (2)polynucleotide 50-30,000 base-pairs.

• The pore size is determined by adjusting the concentration of agarose in a gel (normally in the rank of 0.4-4%)

• Common clinical uses of agarose gel electrophoresis (AGE) are separations of plasma proteins, hemoglobin variants, lipoproteins, and isoenzymes.

Agarose gel preparation Agarose gel preparation

• Gels solution are pored in the gel plate.• A comb with teeth is suspended in the

gel until it cools.• When the gel has cooled and solidified,

pulls the comb out of the gel.• The teeth of the comb leave impressions

in the gel called wells where the sample will be loaded.

• The gel plate is then placed in the electrophoresis chamber.

• Gel acts like a bridge through which the molecules must travel to get from one side of the chamber to the next.

• Polyacrylamide Gels• Polyacrylamide electrophoresis (PAGE) is

performed on a gel formed by polymerizing and cross-linking acrylamides.

• These gels are stronger than agarose gels and also thermostable and transparent.

• In addition to separating fragments by charge and mass, also separates solutes by molecular size.

Polyacrylamide gel Polyacrylamide gel

Types of electrophoresis Lect-2

Types of electrophoresis Lect-2

1. Zone electrophoresisA. Paper electrophoresis - - Protein is easily denatured due to high

absorbance of filter paper. -Works for small peptides and amino acid

separating.B. Thin layer electrophoresis (TLE)C. Gel electrophoresis a. Starch gel electrophoresis b. Polyacrylamide gel electrophoresis

(PAGE) c. Agarose gel electrophoresis

Separation of proteins by paper electrophoresis

Separation of proteins by paper electrophoresis

• Procedure - • In alkaline medium-• Proteins are negatively charged.• They will migrate towards anode

in an electric field.• Separation is based on net

charge and difference in their molecular weight, their motilities are different.

ProcedureProcedure

Immerse a cellulose paper strip into the electrophoresis buffer.

Take it out and evaporate water by drying this paper strip.

Add 3-5 ul of sample on the strip 2 cm far from cathode side electrode.

Put it on electrophoretic device and connect it and electrode with the current device.

• It is carried out at a constant voltage of 80V for 50-60 mints.

• After seperation process, take it out and immersed into fixer solution and than into a dye for 1 mints.

• Than put it into a destaining solution for rinsing.

• Observe the colour and width of the band on the strip to calculate the result.

Gel electrophoresisGel electrophoresis

ElectroendosmosisElectroendosmosis• With a pH 8.0-9.0 used for protein

electrophoresis, proteins take on a negative charge, that is a negative ion cloud forms.

• As the negative ion cloud migrates to the anode, the proteins are pulled to the anode.

• Several gels used routinely for protein electrophoresis, attract positive ions from the buffer and form a positive ion cloud.

• This ion cloud moves in the opposite direction to the cathode.

• This phenomenon is called electro endosmosis or endosmosis.

• The tension created by these oppositely moving ion clouds can affect the movement of sample macromolecules.

• The migration of some proteins can be slowed, some proteins can become immobile, and other proteins are pushed toward the cathode.

• The gamma globulin band in serum, urine, and other body fluids will separate more sharply by being pushed to the cathode.

EndosmosisEndosmosis

• Other important types of electrophoresis

• 2. Isoelectrric focusing

• 3. Peptide mapping

• 4. Western blotting/immunostaining

• 5. Immunoelectrophoresis

• 6. Capillary electrophoresis

• 7. Pulse field gel electrophoresis

• 8. Isotachophoresis

Clinical applications of electrophoresisClinical applications of electrophoresis

• Specific protein electrophoresis is used for quantitative analysis of serum proteins .

• Various classes of Hb can be separated by using this technique.

• It can be used in separation and quantization of major lipoproteins and determination of separate lipid

profile .

•

• It is being used for the separation of whole chromosome .

• Study of CSF protein can be done by electrophoresis.

• It can also be used for analysis of Iso enzyme which are early diagnosis of various disorders of the body. Exampls are -

• Myocardial infarction, heart attack, liver disorders, muscular dystrophy and various type of cancer.

• Creatine kinase (CK), Lactate dehydrogenase (LDH) which is early signal of medical disaster can be estimated by electrophoresis.

• It can be used in diagnosis Multiple myeloma, Liver cirrhosis, Immunodeficiency.

• In addition to that, we can do Western blotting for conformation of AIDS and Southern blotting for prenatal diagnosis of inborn errors, viral infection.