Two-Dimensional Electrophoresis of Proteins using Immobilized pH Gradients

by

Angelika Görg

in collaboration with

Günther Boguth, Alois Harder, Christian Obermaier, Burghardt Scheibe, Robert Wildgruber and Walter Weiss

Technical University of Munich

A LABORATORY MANUAL

http://www.weihenstephan.de/blm/deg

Correspondence Prof. Dr. Angelika Görg

Technical University of Munich

Dept. of Food Technology

D-85350 Freising-Weihenstephan, Germany

http://www.weihenstephan.de/blm/deg

Preface

The current laboratory manual of two-dimensional electrophoresis with immobilized pH gradients (IPG-Dalt) was originally prepared for our own use and for the use by those who came to our laboratory to learn the technique we originally described in Electrophoresis 1988, 9, 531-546.

Moreover, this manual became to be the standard protocol of our one-week courses (GDCh 1990-1997, FEBS 1997), where the participants not only run their own experiments but also their own samples.

Special thanks to Walter Weiss, Günther Boguth, Christian Obermaier, Burghardt Scheibe and Anton Posch for contributing current practice not only to the manual but also to all the courses and work shops we run together all over the world.

Weihenstephan, July 1998 Prof. Dr. Angelika Görg

1 Introduction

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) (O’Farrell 1975), in which proteins are separated according to charge (pI) by isoelectric focusing (IEF) in the first dimension and according to size (Mr) by SDS-PAGE in the second dimension, has a unique capacity for the resolution of complex mixtures of proteins, permitting the simultaneous analysis of hundreds or even thousands of gene products. However, the ecxhange of 2-D gel data between laboratories has been a major problem because of spatial irreproducibility between 2-D gels generated by the conventional method of 2-D PAGE using carrier ampholyte (CA) IEF. Equilibrium CA-IEF cannot be achieved because of pH gradient instability with prolonged focusing time, as the pH gradient moves towards the cathode (‘cathodic drift’) and flattens in the centre (‘plateau phenomenon’). Consequently, time-dependent protein patterns are obtained. In addition, reproducibility of pH gradient profiles is limited by the batch-to-batch variability of CA preparations.

Finally, the problems of pH gradient instability and irreproducibility were overcome by the introduction of immobilized pH gradients (IPG) for IEF (Bjellqvist et al. 1982). IPGs are based on the principle that the pH gradient is generated by a limited number (6-8) of well-defined chemicals (the ‘Immobilines’) which are co-polymerized with the acrylamide matrix. Thus cathodic drift is eliminated, reproducibility enhanced and pattern matching and inter-laboratory comparisons simplified. IPGs allow the generation of pH gradients of any desired range (broad, narrow or ultra-narrow) between pH 3 and 12. Since sample loading capacity of IPG-IEF is also higher than with CA-IEF, especially in combination with narrow (1 pH unit) or ultra-narrow (0.1 pH unit) IPGs, 2D-PAGE with IPGs is the method of choice for micropreparative separation and spot identification.

Two-dimensional electrophoresis with IPGs (IPG-Dalt)

A basic protocol for horizontal as well as vertical two-dimensional electrophoresis with IPGs in the first dimension (IPG-Dalt) was established in 1988 (Görg et al. 1988). Since that time, the protocol has not been changed essentially. Compared to classical 2-D electrophoresis with carrier ampholytes (O’Farrell 1975), the employment of IPG-Dalt has produced significant improvements in 2-D electrophoretic separation, permitting higher resolution, especially with narrow-range IPGs and reproducibility of 2-D patterns both within a laboratory and, more important, between laboratories (Corbett et al. 1994; Blomberg et al. 1995). Moreover, basic proteins (pI >7.5) normally lost by the cathodic drift of carrier ampholyte focusing or separated by NEPHGE (O’Farrell et al. 1977) with limited reproducibility, were perfectly separated under equilibrium conditions using IPGs 4-9, 4-10, and 6-10 for the separation of highly diverse samples such as plant, yeast, mouse liver, human heart, or myeloblast proteins (Görg et al. 1988, 1991, 1993). Recently, very alkaline IPGs up to pH 12 were successfully generated for the 2-D electrophoresis of ribosomal proteins and histones (Görg et al. 1997).

Due to these features, together with the high loading capacity of IPG-Dalt for micro-preparative runs (up to 10 mg of a crude sample preparation can be applied onto a single 2-D gel) (Hanash et al. 1991; Bjellqvist et al. 1993; Posch et al. 1994), IPG-Dalt advanced to the core technology of proteome analysis (Wilkins et al. 1997), facilitating spot identification by peptide mass fingerprinting, MALDI or tandem mass spectrometry, amino acid composition analysis, N-terminal and/or internal peptide microsequencing.

1.2 The protocol of IPG-Dalt

The basic protocol of IPG-Dalt is summarized in Figure 1. The first dimension, isoelectric focusing (IEF), is performed in individual IPG gel strips, 3 mm wide and cast on GelBond PAGfilm (either ready-made Immobiline DryStripsR or laboratory-made, obtained from washed and dried immobiline gels cast by the gradient casting technique of Görg et al. (1980, 1986). Prior to IEF, IPG dry strips are rehydrated to their original thickness of 0.5 mm with a solution containing 8 M urea, 0.5-2% (non-ionic or zwitterionic) detergent, 0.2% dithiothreitol (DTT) and 0.2% carrier ampholytes. The rehydrated strips are then placed onto the cooling plate of an electrofocusing chamber and sample cups are placed onto the surface of the gel strips. Sample entry is critical, and best results are obtained using diluted samples dissolved in 9.5 M urea, 2-4% non-ionic or zwitterionic detergent, 1% DTT, and 0.8% carrier ampholyte (O’Farrell 1975), or -in the case of very hydrophobic proteins- by a mixture of 2M thiourea and 7 M urea instead of 9.5M urea and/or other detergents (Rabilloud et al. 1997). For better sample entry, a low voltage gradient is applied across the gel for the first hour. Voltage is then increased to 3500 V (Multiphor) (Görg et al. 1988), or even up to 8000 V (IPGphor) until the steady state with constant focusing patterns is obtained.

As an alternative to cup-loading, samples can also be applied by in-gel rehydration (Rabilloud et al. 1994). The latter procedure may be especially advantageous for high sample loads such as for micropreparative 2D-PAGE.

An ecxiting new development for simplification of IPG-IEF is the introduction of an integrated system (IPGphor) where in-gel rehydration and IEF are performed in one step overnight, without personal assistance (Islam et al. 1998).

Whatever system is used for isoelectric focusing, after IEF to the steady state, the IPG strips are equilibrated in presence of SDS, DTT, urea, glycerol and iodoacetamide (IAA), and then placed onto the surface of a horizontal or on top of a vertical SDS gel. Alter-native procedures using tributylphosphine instead of DTT and IAA have been success-fully applied for hydrophobic proteins (e.g. wool filament proteins) (Herbert et al. 1998).

For horizontal set-ups, the laboratory-made or ready-made SDS-PAGE gel (EcxelGel SDS), cast on plastic backing, is placed onto the cooling plate of a horizontal electro-phoresis system, and the equilibrated IPG gel strip is transferred gel-side-down onto the surface of the the SDS gel alongside the cathodic electrode wick or buffer strip.

For vertical setups, the equilibrated IPG gel strips are loaded on top of vertical SDS poly-acrylamide gels, with or without embedding in agarose. Vertical setups are especially useful for multiple runs (up to 20 at a time) (Anderson and Anderson, 1978).

Upon completion of electrophoresis, the polypeptides are either stained with Coomassie Brilliant Blue or silver nitrate, or detected by fluorescence or autoradiography. Alter-natively, proteins are transferred ("blotted") onto an immobilizing membrane and detected with specific reagents such as antibodies.

For spot identification, the spots are ecxised from the gel or the blotting membrane and subjected directly or after (enzymatic or chemical) cleavage to Edman degradation, amino acid composition analysis, or mass spectrometry.

Legend to Figure 1

(A) Assembly of the polymerisation cassette for the preparation of IPG and SDS gels
on plastic backings (Glass plates, GelBond PAGfilm and 0.5 mm thick U-frame)

(B) Casting of IPG- and/or pore gradient gels

(C) Cutting of washed and dried IPG slab gels (or Immobiline DryPlates) into individual IPG
strips

(D) Rehydration of individual IPG strips in a vertical rehydration cassette,

(E) in the reswelling tray, or

(F) in the IPG strip holder (IPGphor)

(G) IEF in individual IPG gel strips directly on the cooling plate of the IEF chamber,

(H) in the DryStrip kit, or

(I) on the IPGphor

(K) Storage of IPG strips after IEF

(L) Equilibration of IPG strips prior to SDS-PAGE

(M) Transfer of the equilibrated IPG strip onto the surface of the laboratory-made
horizontal SDS gel along the cathodic electrode wick, or

(N) onto the surface of a ready-made horizontal SDS gel along the cathodic buffer strip

(O) Loading of the equilibrated IPG gel strip onto the surface of a vertical SDS gel

Fig 1. Procedure of IPG-Dalt based on the protocol of Görg et al. (1988)