Protein Processing in Food and Bioproduct Manufacturing and Techniques for Analysis - Food and Industrial Bioproducts and Bioprocessing - page 101

with a second property in the orthogonal direction (Pastorello and Trambaioli, 2001).
2D electrophoresis is a powerful method for the analysis of complex protein mixtures
extracted from cells, tissues, or other biological samples, and can resolve hundreds to
thousands of individual protein species detected as spots or bands after staining (Friedman
et al ., 2009 ).

Native Polyacrylamide Gel Electrophoresis (PAGE)
PAGE under native conditions has been used to separate soluble proteins which retain their
biological and enzymatic properties. Factors affecting the migration of proteins in native
PAGE are: size, shape, and native charge (Dunn, 1989). A derivative method from native
PAGE developed by Schägger and Von Jagow (1991), known as Blue Native PAGE, was
originally used for the qualitative and quantitative analysis of mitochondrial protein complexes
and proteins ranging in molecular weights from 10 to 10 000 kDa (Dresler et al ., 2010 ).

SDS-PAGE
Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures proteins through
specific binding to the hydrophobic tail around the polypeptide backbone, giving a net uni-
form negative charge per molecule (Dunn, 1989). The binding of SDS unfolds the protein
as a consequence of hydrogen bond cleavage, and blockage of hydrophobic interactions.
Consequently, electrophoretic separation is only dependent on the molecular weight and
proteins migrate in the anodal direction. Moreover, proteins can be denatured by the addi-
tion of strong reducing agents, such as dithiothreitol (DTT) or -mercaptoethanol, which

disrupt disulfide bonds between cysteine residues, allowing a more detailed characterization
of proteins (Dunn, 1989 ).
Lately, the discontinuous Laemmli system buffer (containing 0.1% w/v SDS) is most
commonly used for SDS-PAGE. The method has been widely used for the characterization
of the protein profiles of processed whey, meat, cereal, legume, and seed proteins (Roy
et al ., 2007 ; Miyamoto et al ., 2009 ; Boye et al ., 2010 b; Sikes et al ., 2010 ; Warchalewski and
Gralik, 2010) as well as the analysis of membrane proteins (Braun et al ., 2009 ). Further
information on SDS-PAGE electrophoresis can be found elsewhere (Amersham, 1999a).

β

Immunoelectrophoresis
Immunoelectrophoresis is a specific method characterized by the presence of polyclonal
monospecific antibodies against a specific protein in the agarose gel. The running buffer
used in this method should have a pH corresponding to the isoelectric point of the antibody.
The fixation of the latter in the gel leads to the formation of a precipitate comprised of the
antigen and the specific antibody, which may be visualized by using a Coomassie blue stain
or some other suitable technique (Westermeier and Scheibe, 2009). Immunoelectrophoresis
has been used for the qualitative and quantitative analysis of individual proteins in complex
mixture (Hansen and Larsen, 2008).

Isoelectric focusing
Isoelectric focusing (IEF) is an electrophoretic method that separates proteins based on their
isoelectric points (pI, neutral charge state). The migration is performed in the presence of a
continuous pH gradient generated using carrier ampholytes or immobilized pH gradients
gels (Dunn, 1989; Amersham, 1999a). After migration according to their charge, proteins
attain a steady sate when they reach the pH value corresponding to their pI. The technique
is commonly used in combination with SDS-PAGE in 2D electrophoresis. IEF can also be
performed under native conditions (Dunn, 1989; Friedman et al ., 2009 ).