Electrophoresis part 3
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This document discusses two electrophoresis techniques: capillary electrophoresis and isoelectric focusing. Capillary electrophoresis uses very small volumes of samples and reagents in fused silica capillaries, allowing for high resolution, automation, and detection of low analyte levels. Isoelectric focusing separates proteins based on their isoelectric point, where they have no net charge and become immobilized in a pH gradient gel. Both techniques have various applications in biochemistry including separating proteins, DNA, amino acids, and other biomolecules.
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Electrophoresis part 3
- 1. ELECTROPHORESIS- Part 3 1.Capillary Electrophoresis 2.Isoelectric Foccusing Dr.Rittu Chandel Junior Resident (3rd Yr),MD Biochemistry Grant Government Medical College, Mumbai -400008 07-08-2014
- 2. 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 2Dr.Rittu Chandel
- 3. 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 3Dr.Rittu Chandel This eliminates zone broadening and convection
- 4. 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.
- 6. SAMPLE INJECTION • High Voltage Injection/Electrokinetic Injection • High Pressure Injection/Hydrodynamic injection 6Dr.Rittu Chandel
- 7. This is where the Electroosmotic flow What happens to the + cations when we turn on the power?
- 8. 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
- 10. pH, Silanol Population, and the rate of EOF flow. 0 2 4 6 8 10 12 14 16 18 20 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.
- 13. APPLICATIONS • Separation of 1.Amino acids 2.Peptides 3.Proteins 4.DNA fragments 5.Nucleic Acids 6.Vitamins 7.Porphyrins 8.Carbohydrates 13Dr.Rittu Chandel
- 14. 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. Netcharge -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 < pI Positive charge NH3 + COO- NH3 + COO- pH = pI NH2 COO- NH2 COO- pH > pI Negative charge
- 15. 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 15Dr.Rittu Chandel
- 16. Traditional Equipment for Isoelectric focusing (IEF): Ampholytes polyacrylamide Cathode (-) electrode solution Anode (+) electrode solution
- 17. 17Dr.Rittu Chandel Stable pH gradient
- 20. PREPARATION OF IEF GEL Gel is polymerised Second glass plate is placed on first Mixture is poured over a glass plate which contain spacer Carrier ampholytes (suitable pH) and riboflavin mixed with acrylamide solution
- 21. Ampholytes form a pH gradient between anode and cathode Potential difference is applied Electrode wicks are laid along the long length of each side of the gel After the gel has set glass plates are prised apart This takes 2-3 hr
- 22. Become stationary when they reaches isoelectric point Proteins having positive charge will migrates towards the cathode. negatively charged protein will migrates towards anode Voltage is again applied for 30 min Samples applied by laying on gel filter paper soaked in the sample The power is then turned off
- 23. Destained Gel is stained with Coomasie Brilliant Blue This precipitaes the proteins and allows smaller ampholytes to be washed out The gel is washed with trichloroacetic acid
- 25. IMMOBILIZED pH Gradient Strip Dr.Rittu Chandel 25
- 26. IPGphor (IEF System) Amersham Pharmacia Biotech Inc. Protein IEF Cell Bio-Rad Laboratories Equipment for Isoelectric focusing (IEF):
- 27. 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 27Dr.Rittu Chandel