Two-Dimensional Separation of Protein Samples

Separation of labeled proteins is carried out using traditional 2-D polyacrylamide gel electrophoresis (chapters 2–4: EF using Ettan™ IPGphor™ 3 Isoelectric Focusing System and accessories; Electrophoresis conditions with precast gels for both Ettan™ DALTsix and Ettan™ DALTtwelve; Running a MultiPhor™ II protocol).

Protocol

A. Combining protein samples for multiplexing

Protein samples labeled with different CyDye™ DIGE Fluor minimal dyes are combined according to the experimental design. For best results, one or two labeled protein samples (usually Cy3 or Cy5) are combined with a labeled internal standard (usually Cy2), which is a pool of aliquots of all biological samples in the experiment.

B. Diluting labeled protein sample in sample buffer

The sample mixture is diluted further in sample buffer prior to separating the individual proteins on a 2-D gel.

Ettan™ DIGE system-compatible 2× sample buffer contains 7 M urea, 2 M thiourea, 2% CHAPS (w/v), 2% IPG buffer or Pharmalyte^® (v/v) for IEF, 2% DTT (w/v).

Add 1 volume of 2× sample buffer to sample. Mix and leave on ice for at least 10 min.

C. Rehydrating Immobiline® DryStrip gel

Refer to sections 2.4–2.7 for a discussion of rehydration and sample application methods.

Ettan™ DIGE system-compatible rehydration buffer contains 7 M urea, 2 M thiourea, 4% CHAPS (w/v), 1% IPG buffer or Pharmalyte^® (v/v) for IEF, 2% (w/v) DTT.

D. Separating proteins in the first dimension

Ettan™ IPGphor™ 3 Isoelectric Focusing System and MultiPhor™ II Electrophoresis System are both suitable for Ettan™ DIGE system applications. Detailed instructions for use of the systems are given in chapters 2 (IEF using Ettan™ IPGphor™ 3 Isoelectric Focusing System and accessories) and 4 (First-dimension IEF using MultiPhor™ II Electrophoresis System and Immobiline^® DryStrip Kit), respectively. Ettan™ DIGE applications are described in detail in the Ettan™ DIGE user manual.

E. Separating proteins in the second dimension

Low-fluorescence glass plates must be used for gels used in Ettan™ DIGE system. Standard glass or plastic-backed plates can result in the generation of a high background signal.

DIGE Gel is a 12.5% precast, low-fluorescent polyacrylamide gel cast in a low-fluorescent glass cassette specially developed for 2-D DIGE analysis. DIGE Gel should be used with the DIGE Buffer Kit, which consists of concentrated running buffers and Sealing Solution for attaching Immobiline® DryStrip Gels (IPG strips) to the top of the polyacrylamide gel. The capacity of the buffer system is similar to the commonly used Laemmli (Tris-Glycine) buffer system, and the separation performance of DIGE Gel is comparable to 12.5% Laemmli gels.

DALT gels are large enough to accommodate the longest Immobiline^® DryStrip gels (24 cm) and can be run in batches of up to 12 gels at a time. DALT gels can also be poured using the DALT gel caster. DALT gels can also be run using the DIGE buffer kit. Detailed protocols for gel casting can be found in the Ettan™ DIGE user manual, Ettan™ DALTtwelve user manual, and Ettan™ DALTsix user manual.

The procedures for equilibrating strips, positioning them, and electrophoresis are identical to those for standard 2-D analysis. Refer to sections 3.1 (Equilibrating Immobiline^® DryStrip gels) and 3.3 for details.

If the gels are to be scanned immediately, store the gels in SDS electrophoresis running buffer at room temperature in a light-tight container. Scan the gels as soon as possible as the protein spots in the gel will diffuse with time. If the gels cannot be scanned on the day of running, they should be stored in the dark at +4 °C and kept moist. Remember to let the gels warm to room temperature before scanning because temperature affects the fluorescent signal.

Do not fix the gels prior to scanning as this will affect quantitation of the labeled protein spots.