ELECTROPHORESIS

Charge Separa

tion

Size Separa

tion

Analyze

Identify

Purify

Mixture of

Charged Molecule

s

Positive Molecules

Negative Molecules

Presented By,PRADEEP S.

JAISWAL.MSC PART-2.SEMESTER-4PAPER-1

CONTENTS :•Principle of electrophoresis.•Factor affecting electrophoresis.•Support media.•Technique.•Detection and quantification.•Types of electrophoresis.•Application of electrophoresis technique.•Advantage.•Disadvantage.•References.

Principle of Electrophoresis• Positive & negative electrical charges are frequently

associated with biomolecules. • Electrophoresis of positively charged particles (cations) is

called cataphoresis, while electrophoresis of negatively charged particles (anions) is called anaphoresis. • When they placed in electric field , charged biomolecules

move towards the electrodes of opposite charge due to the phenomenon of electrostatic attraction.• An ampholyte become positively charged in acidic

conditions & migrate to cathode, In alkaline conditions they become negatively charge & migrate to anode.

Factors affecting Electrophoresis

Inherent Factors:• Magnitude of its charge.• Charge density.• Molecular weight.

External Environmental Factors:• Solution Ph.• Electric Field.• Solution Viscosity.• Temperature.

•Starch Gel:Separate macromolecules on basis of surface charge and molecular size.•Cellulose Acetate:Hardly used in routine clinical application.•Agarose:Used in AGE for the separation of Serum, Urine or CSF proteins, Hb variants, Isoenzymes, Lipoproteins etc.•Polyacrylamide:Pore size does not allow larger proteins like fibrinogen, B1-lipoprotiens, Y-globulins etc, to migrate•Separation is based on both charge-to-mass ratio and molecular sieving .•Buffers: in gel electrophoresis are used to provide ions that carry a current & maintain the pH at a relatively constant value.

Support Media:

• Submerging the Gel: First, a salty solution is poured over the gel in order to conduct electricity.Loading the Samples: The DNA samples, colored with blue loading dye, are placed in the slots made in the agarose gel.

• Electrophoretic run:The current is switched on after the sample has been applied to the paper & the paper has been equilibrated with the buffer ,Smaller biomolecules travel farther down the gel, while lager ones remain closer to the point of origin.

Technique:

Detection & Quantification:

• Optial method: UV absorption by separated components Proteins: Absorb UV (230-280nm).After the gel has been run, the ethidium bromide that was added to it causes the gel to glow under ultraviolet light.

• Staining: By reacting separated components with a stain or dye and detecting by absorption or fluorescence .Eg. Ninhydrin used for staining of amino acid , peptides, and proteins.

Types of Electrophoresis

1. Zone Electrophoresis.2. Slab gel Electrophoresis.3. Disc Electrophoresis.4. Isoelectric Focusing Electrophoresis.5. 2 Dimensional Electrophoresis.6. Capillary Electrophoresis.7. Microchip Electrophoresis.

Zone Electrophoresis

• Produce zone of proteins that are heterogeneous & physically separated from one another.• Classified according to

type & structure of the support material e.g. AGE, CAE, PAGE etc.

Slab Gel Electrophoresis:• It is primary method used in

clinical chemistry lab.• It has ability to simultaneously

separate several samples in one run.• It uses a rectangular gel

regardless of thickness.• Gels are cast on sheets of

plastic backing.• It is useful in separation of

serum proteins, isoenzymes, lipoproteins, hemoglobin & fragments of DNA & RNA.

• The bands observed can be compared to those of the known 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.

• If several mixtures have initially been injected next to each other, they will run parallel in individual lanes.

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Gel Electrophoresis

Disc Electrophoresis3-gel system:• Small-pore separating gel (running gel).• A larger-pore spacer gel (stacking gel).• And a thin layer of large-pore monomer

solution (sample gel) .• The different composition cause

discontinuities in the electrophoresis matrix.• During electrophoresis, all proteins migrate

easily through the large-pore gels and stack up on the separation gel in a very thin zone.• This improves resolution and concentrates

protein components at the border (or starting zone).

Isoelectric Focusing Electrophoresis:

• It is separation method that resolves proteins markers on the basis of their isoelectric points.• Proteins migrate through a

zone in a medium where the pH of the gel matches its PI.• At this point, the charge of

the protein becomes zero & its migration ceases. It becomes focused.• A high voltage power source

is needed because carrier ampholytes are used in relatively high concentrations. Thus it must be cooled.

2-Dimensional Electrophoresis

• This technique combines the techniques of IEF which separates proteins in a mixture according to charge (PI) with the size separation technique of SDS-PAGE.

• 1st Dimension – Charge dependent IEP .

• 2nd Dimension – Molecular weight dependent electrophoresis

• It achieves the highest resolving power for the separation of DNA fragments.

Capillary Electrophoresis• Separation in narrow bore fused

silica capillaries filled with buffer.• Sample is loaded after filling

capillary with buffer & electric field applied.• Electro-osmotic flow (EOF)

controls the amount of time solute remain in the capillary.• Cations migrate fastest due to

EOF & electrophoretic attraction towards the cathodes.• Anions move slower because EOF

is slightly greater than the attraction towards the anode & repulsion from cathode.

Microchip Electrophoresis:

• Similar in principle to CE, but differs in that the separation channels, sample injection channels and reservoirs are all fabricated into the same planar substrate using photolithographic processes.• More so, sample preparationand/or

precolumn or postcolumn reactors, detectors, and excitation sources are intergrated into the chip.

APPLICATIONS OF ELECTROPHORESIS1. DNA Sequencing .

2. Medical Research.

3. Protein research/purification.

4. Agricultural testing.

5. Separation of organic acid, alkaloids, carbohydrates, amino acids,

alcohols, phenols, nucleic acids, insulin.

6. In food industry .

8. Electrophoresis in combination with autoradiography is used to

study the binding of iron to serum proteins.

9. Used for analysis of terpenoids , steroids and antibiotics.

10. For testing purity of thyroid hormones by zone electrophoresis.17

Advantages

• Provides a clear link between similar results.• Relatively simple to perform.• Can test DNA from any type of evidence (hair, blood,

skin, etc.).• Relatively inexpensive.Disadvantages

•The gel can be altered and provide inaccurate results.

•User error can be catastrophic, depending on the mistake.

References

• Electrophoresis- New york: John Wiley and Sons .