2D gele electrophoresis for proteomics tutorial

A tutorial by Dr James R. Jefferies ( Parasitology Group, Institute of Biological Sciences, University of Wales at Aberystwyth).

Content:

1. What is 2D Gel Electrophoresis?
2. What is it used for?
3. How is it Performed?
4. How is the pH Gradient Formed for the IEF Procedure?
1. Ampholytes.
2. Immobilines.
5. Immobilized pH Gradients.
1. Why use immobilized pH gradients for IEF?
2. Disadvantages of IPGs.
6. 2D Procedure.
1. 1st dimension (IEF).
2. 2nd dimension (SDS-PAGE).
7. Sample Preparation.
1. Pre-prep sample considerations.
2. Contaminated?
3. What sort of sample?
4. What does it contain?
5. Are there proteases?
6. Is it precious?
7. Whole organism or just a bit?
8. Individual organelles?
9. Protein pre-fractionation?
10. Mechanical tissue disruption.
8. Sample Solubilisation.
1. Ultracentrifugation.
2. SD buffer components.
9. Chaotrophe.
10. Reductants.
11. Detergents-Surfactants.
12. Ampholytes.
13. Interfering Substances.
1. Lipids.
2. Proteases.
3. Nucleic acids.
4. Polysaccharides.
5. Salts.
14. Protein Estimation.
15. Gel Electrophoresis.
1. IEF strips.
16. IEF Run.
17. Strip Equilibration.
18. SDS-PAGE Run.
19. Staining.
1. Zinc or copper staining.
2. Coomassie Blue staining.
3. Silver staining.
4. Fluorescent stains.
20. General Considerations.
21. References.

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Last update 25-Jul-2006, Rating n/a of 0 votes.