Table of Contents
1 Introduction
Two-dimensional electrophoresis with IPGs (IPG-Dalt)
1.2 The protocol of IPG-Dalt
2 Sample preparation
2.1 General aspects
2.2 Chemicals
2.3 Buffers
2.4 Procedure
3 Casting of IPG and SDS gels
3.1 Preparation of Immobiline (IPG) gels
3.1.1 Apparatus and chemicals
3.1.2 Reagent solutions
3.1.3 IPG Gel casting
3.2 Preparation of SDS gels
3.2.1 Apparatus and chemicals
3.2.2 Reagent solutions
3.2.2.1 Reagent solutions for horizontal SDS gels
3.2.2.2 Reagent solutions for vertical SDS gels
3.2.3 SDS Gel casting procedure
3.2.3.1 Horizontal SDS pore gradient gels on plastic backing
3.2.3.2 Vertical SDS gels for multiple runs
4 First dimension: IEF in individual IPG strips
4.1 Apparatus and chemicals
4.2 Reagent solutions
4.3 Running IPG strips
4.3.1 IEF following sample cup loading
4.3.1.1 Rehydration of IPG strips
4.3.1.2 Analytical IEF for multiple runs
4.3.1.3 Analytical and micropreparative IEF using Pharmacia's DryStrip Kit
4.3.2 IEF following in-gel rehydration of sample
4.3.3 IEF with IPGphor
5 Equilibration of the IPG gel strips
5.1 Apparatus and Chemicals
5.2 Buffer solutions
5.3 Procedure
6 Second dimension: SDS-PAGE
6.1 Apparatus and chemicals
6.2 Reagent solutions
6.2.1 Reagent solutions for horizontal SDS gels
6.2.2 Reagent solutions for vertical SDS gels
6.3 Running SDS gels
6.3.1 Horizontal SDS-PAGE
6.3.1.1 SDS-PAGE on laboratory-made gels
6.3.1.2 SDS-PAGE on ready-made EcxelGel
6.3.2 Vertical SDS-PAGE
6.4 Fixing
7 Visualization
7.1 Silver staining protocol according to Blum et al. (1987) (mod.)
7.2 Silver staining protocol according to Heukeshoven and Dernick (1988) (modified)
7.3 Silver staining protocol according to Oakely et al. (1980) (mod.)
7.4 Gel drying
8 Blotting
8.1 Reagent solutions
8.2 Procedure
8.3 Visualization of the blotted proteins by Coomassie blue staining
9 Microcharacterization of protein spots
10 Pitfalls
11 Original Protocol of IPG-Dalt
12 References
