ELECTROPHORESIS- Part 3 1.Capillary Electrophoresis

2.Isoelectric Foccusing

Dr.Rittu ChandelJunior Resident (3rd Yr),MD Biochemistry

Grant Government Medical College, Mumbai -40000807-08-2014

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CAPILLARY ELECTROPHORESIS

• High Performance Capillary Electrophoresis/ Capillary Zone Electrophoresis/ Free Solution Capillary Electrophoresis

• Microlitres of reagent used• Nanolitres of sample required

• Online detection to femtomole sensitivity• Ease of automation• Less time consuming

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CAPILLARY ELECTRPHORESIS

Capillary made of Fused silica, thin exterior covering of polyimide

Inner diameter – 10-100 μm

Outer diameter- 300 μm

Length – 50-100 cm

Voltage - 10-50 KV

Large surface to volume ratio, provides enhanced heat dissipation

This eliminates zone broadening and convection

Anode+

Cathode-

Detector

The interior wall of the capillary contains charged sites that are created by the ionization of Silanol ( Si-OH) groups on the fused silica.

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CAPILLARY ELECTROPHORESIS

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SAMPLE INJECTION

• High Voltage Injection/Electrokinetic Injection

• High Pressure Injection/Hydrodynamic injection

This is where the Electroosmotic flow

What happens to the + cations when we turn on the power?

Analytes with a net positive charge will move faster than EOF

EOF

Analytes with no net charge will move at the same speed as the EOF.(This is a useful tool that helps us to measure the EOF.)

EOF

Analytes with a net negative charge will move slower than EOF

EOF

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pH, Silanol Population, and the rate of EOF flow.

02468101214161820

2 5 8 11

EOF

• At very low pH, not many silanols are ionized and the EOF is slow.

• As pH increases the number of ionized sites also increases. The EOF speed rises steadily.

• At very high pH values, a maximum number of ionized sites is reached. The EOF speed also reaches a maximum.

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APPLICATIONS

• Separation of

1.Amino acids

2.Peptides

3.Proteins

4.DNA fragments

5.Nucleic Acids

6.Vitamins

7.Porphyrins

8.Carbohydrates

Isoelectric point (pI): Isoelectric point is the pH of a solution at which the net charge of protein is

zero. In electrophoresis there is no motion of the particles in an electric

field at the isoelectric point.

Net

cha

rge

-3

-2

-1

0

1

2

3

2 3 4 5 6 7 8 9 10 11

pH

Isoelectric point

NH3+

COOH

NH3+

COOH

pH < pIPositive charge

NH3+

COO-

NH3+

COO-

pH = pI

NH2

COO-

NH2

COO-

pH > pINegative charge

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ISOELECTRIC FOCUSSING

• Immobilization of molecules at isoelectric pH during electrophoresis

• Very high resolution technique for protein

• Creation of pH gradient- low molecular mass (600-900 D) oligomers bearing aliphatic amino acids and carboxylic acid groups

Traditional Equipment for Isoelectric focusing (IEF):

Ampholytespolyacrylamide

Cathode (-) electrode solution

Anode (+) electrode solution

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Stable pH gradient

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PREPARATION OF IEF GEL

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IMMOBILIZED pH Gradient Strip

IPGphor (IEF System)Amersham Pharmacia Biotech Inc.

Protein IEF CellBio-Rad Laboratories

Equipment for Isoelectric focusing (IEF):

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APPLICATIONS

• 2D gel electrophoresis• Phenotyping of α1 antitrypsin• Genetic variants of enzymes, hemoglobin, other

proteins• Separating isoenzymes- Creatine Kinase,Alkaline

Phospatase, Acid Phosphatase• In forensic science• Oligoclonal Ig band in CSF

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THANK YOU