electrophoresis part 3
TRANSCRIPT
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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
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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
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This is where the Electroosmotic flow
What happens to the + cations when we turn on the power?
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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
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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
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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
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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