Food Biochemistry and Food Processing (2 edition)

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22 Application of Proteomics to Fish Processing and Quality 409

remains essentially as outlined earlier. In the following sections,

a general protocol is outlined briefly with some notes of special

relevance to the seafood scientist. For more detailed, up-to-date

protocols, the reader is referred to any of a number of excellent

reviews and laboratory manuals such as Berkelman and Stenst-

edt (1998), Gorg et al. (2000, 2004), Kraj and Silberring (2008), ̈

Link (1999), Simpson (2003), Walker (2005) and Westermeier

and Naven (2002).

Sample Extraction and Cleanup

For most applications, sample treatment prior to electrophoresis

should be minimal in order to minimize in-sample proteolysis

and other sources of experimental artifacts. We have found direct

extraction into the gel reswelling buffer (7-M urea, 2-M thiourea,

4% (w/v) CHAPS [3-(3-chloramidopropyl)dimethylamino-

1-propanesulfonate], 0.3% (w/v) DTT [dithiothreitol], 0,5%

Pharmalyte ampholytes for the appropriate pH range), sup-

plemented with a protease inhibitor cocktail, to give good

results for proteome extraction from whole Atlantic cod larvae

(Guðmundsdottir and Sveinsd ́ ́ottir 2006, Sveinsdottir et al. ́

2008) and Arctic charr (Salvelinus alpinus) liver (Coe and

Vilhelmsson 2008). Thorough homogenization is essential to

ensure complete and reproducible extraction of the proteome.

Cleanup of samples using commercial two-dimensional sample

cleanup kits may be beneficial for some sample types.

First-Dimension Electrophoresis

The extracted proteins are first separated by IEF, which is most

conveniently performed using commercial dry IPG gel strips.

These strips consist of a dried IPG-containing polyacrylamide

gel on a plastic backing. Ready-made IPG strips are currently

available in a variety of linear and sigmoidal pH ranges. This

method is thus suitable for most 2DE applications and has all

but completely replaced the older and less reproducible method

of IEF by carrier ampholytes in tube gels. Broad-range linear

strips (e.g., pH 3–10) are commonly used for whole-proteome

analysis of tissue samples, but for many applications narrow-

range and/or sigmoidal IPG strips may be more appropriate as

these will give better resolution of proteins in the fairly crowded

pI 4–7 range. Narrow-range strips also allow for higher sample

loads (since part of the sample will run off the gel) and thus may

yield improved detection of low-abundance proteins.

Before electrophoresis, the dried gel needs to be reswelled to

its original volume. A recipe for a typical reswelling buffer is

presented earlier. Reswelling is normally performed overnight

at 4◦C. Application of a low-voltage current may speed up the

reswelling process. Optimal conditions for reswelling are nor-

mally provided by the IPG strip manufacturer. If the protein

sample is to be applied during the reswelling process, extraction

directly into the reswelling buffer is recommended.

IEF is normally performed for several hours at high voltage

and low current. Typically, the starting voltage is about 150

V, which is then increased step-wise to about 3500 V, usually

totaling about 10,000 to 30,000 Vh, although this will depend

on the IPG gradient and the length of the strip. The appropriate

IEF protocol will depend not only on the sample and IPG strip,

but also on the equipment used. The manufacturer’s instructions

should be followed. Gorg et al. (2000) reviewed IEF for 2DE ̈

applications.

Equilibration

Before the isoelectrofocused gel strip can be applied to the

second-dimension slab gel, it needs to be equilibrated for 30–45

minutes in a buffer containing SDS and a reducing agent such as

DTT. During the equilibration step, the SDS–polypeptide com-

plex that affords protein-size-based separation will form and the

reducing agent will preserve the reduced state of the proteins. A

tracking dye for the second electrophoresis step is also normally

added at this point. A typical equilibration-buffer recipe is as

follows: 50 mM Tris-HCl at pH 8.8, 6-M urea, 30% glycerol,

2% SDS, 1% DTT, and trace amount of bromophenol blue. A

second equilibration step in the presence of 2.5% iodoacetamide

and without DTT (otherwise identical buffer) may be required

for some applications. This will alkylate thiol groups and prevent

their reoxidation during electrophoresis, thus reducing vertical

streaking (Gorg et al. 1987). ̈

Second-Dimension Electrophoresis

Once the gel strip has been equilibrated, it is applied to the

top edge of an SDS-PAGE slab gel and cemented in place us-

ing a molten agarose solution. Optimal pore size depends on

the size of the target proteins, but for most applications gra-

dient gels or gels of about 10% or 12% polyacrylamide are

appropriate. Ready-made gels suitable for analytical 2DE are

available commercially. While some reviewers recommend al-

ternative buffer systems (Walsh and Herbert 1999), the Laemmli

method (Laemmli 1970), using glycine as the trailing ion and

the same buffer (25-mM Tris, 192-mM glycine, 0.1% SDS) at

both electrodes, remains the most popular one. The gel is run

at a constant current of 25 mA until the bromophenol blue dye

front has reached the bottom of the gel.

Staining

Visualization of proteins spots is commonly achieved through

staining with colloidal Coomassie Blue G-250 due to its low

cost and ease of use. A typical staining procedure includes fixing

the gel for several hours in 50% ethanol/2%ortho-phosphoric

acid, followed by several 30-minute washing steps in water, fol-

lowed by incubation for 1 hour in 17% ammonium sulfate/34%

methanol/2%ortho-phosphoric acid, followed by staining for

several days in 0.1% Coomassie Blue G-250/17% ammonium

sulfate/34% methanol/2%ortho-phosphoric acid, followed by

destaining for several hours in water. There are, however, com-

mercially available colloidal Coomassie staining kits that do not

require fixation or destaining.

A great many alternative visualization methods are available,

many of which are more sensitive than colloidal Coomassie

and thus may be more suitable for applications where visual-

ization of low-abundance proteins is important. These include