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principle of antibodies binding specifically to antigens in biological tissues. The
antibody-antigen binding can be visualized after immunohistochemical staining by
a fluorescent dye, radioactive tracer, colloidal gold particle, enzyme such as
Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP). This technique can
be performed on formaldehyde fixed and paraffin-embedded (FFPE) tissues allowing access to archival material. A key advantage of IHC is that it not only allows
analysis of the anatomy of the tissue of interest but also visualization of the spatial
distribution and expression of specific antigens or cellular components in a variety
of tissue sections. Immunohistochemical analysis of soy protein allows the detection of all soy protein with all forms of soy additives (texturates, concentrates, soy
flour, isolates) with appropriate epitomes (Pospiech et al. 2009).
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Electrophoresis is a phenomenon that describes migration of charged particles
towards opposite electrode under the influence of electric field. This technique finds
wide application in separating proteins according to their electrophoretic mobility
which depends on charge, molecule size and structure of the proteins. Electrophoresis
in polyacrylamide gels (PAG) is frequently referred to as gel electrophoresis
(PAGE). PAG is a three-dimensional mesh network polymer formed from polymerisation of acrylamide monomer and a cross-linker, bis-acrylamide under the catalyzation of ammonium per-sulphate and base tetra methylenediamine (TEMED). PAG
is a versatile supporting matrix because of its stability with little adsorption and
electro-osmosis effect provided by its neutrally charged nature. Acrylamide gels
with low gel percentage (10–40%) have small pore size. They are used in techniques
such as SDS-PAGE to separate proteins according to their size and to identify their
relative molecular mass.
Two different buffer systems, continuous and discontinuous, can be used in electrophoresis. In continuous system, only one separating gel is used with same buffer
in the tanks and the gel. Whereas in the discontinuous system two-sided gel preparation with different buffers is used (Buyukkoroglu et al. 2018).
To prepare protein samples for SDS-PAGE, they are first boiled in buffer solution
containing an anionic detergent (sodium dodecyl sulphate) that denatures the protein secondary and tertiary structures and opens up into a rod-shaped structure as
well as gives negative charge to the molecule and a strong reducing agent such as
mercaptoethanol and Dithiothreitol (DTT) that could disrupt any disulfide bridge
holding protein tertiary structures. The buffer solution along with protein sample at
an appropriate pH is loaded onto the top of stacking gel with large pore size to concentrate the protein into narrow bands prior to their entry into resolving gel of
smaller pore size. Stacking gel is poured on top of the resolving gel and it is into this
gel that the wells are formed. On applying electric field, a voltage gradient is formed
between the chloride (high negative charge) and glycerine ions (low negative
charge) in the electrode buffer at pH 6.8, which serves to stack the proteins into
Recent Advances in Analysis of Food Proteins
principle of antibodies binding specifically to antigens in biological tissues. The
antibody-antigen binding can be visualized after immunohistochemical staining by
a fluorescent dye, radioactive tracer, colloidal gold particle, enzyme such as
Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP). This technique can
be performed on formaldehyde fixed and paraffin-embedded (FFPE) tissues allowing access to archival material. A key advantage of IHC is that it not only allows
analysis of the anatomy of the tissue of interest but also visualization of the spatial
distribution and expression of specific antigens or cellular components in a variety
of tissue sections. Immunohistochemical analysis of soy protein allows the detection of all soy protein with all forms of soy additives (texturates, concentrates, soy
flour, isolates) with appropriate epitomes (Pospiech et al. 2009).
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Electrophoresis is a phenomenon that describes migration of charged particles
towards opposite electrode under the influence of electric field. This technique finds
wide application in separating proteins according to their electrophoretic mobility
which depends on charge, molecule size and structure of the proteins. Electrophoresis
in polyacrylamide gels (PAG) is frequently referred to as gel electrophoresis
(PAGE). PAG is a three-dimensional mesh network polymer formed from polymerisation of acrylamide monomer and a cross-linker, bis-acrylamide under the catalyzation of ammonium per-sulphate and base tetra methylenediamine (TEMED). PAG
is a versatile supporting matrix because of its stability with little adsorption and
electro-osmosis effect provided by its neutrally charged nature. Acrylamide gels
with low gel percentage (10–40%) have small pore size. They are used in techniques
such as SDS-PAGE to separate proteins according to their size and to identify their
relative molecular mass.
Two different buffer systems, continuous and discontinuous, can be used in electrophoresis. In continuous system, only one separating gel is used with same buffer
in the tanks and the gel. Whereas in the discontinuous system two-sided gel preparation with different buffers is used (Buyukkoroglu et al. 2018).
To prepare protein samples for SDS-PAGE, they are first boiled in buffer solution
containing an anionic detergent (sodium dodecyl sulphate) that denatures the protein secondary and tertiary structures and opens up into a rod-shaped structure as
well as gives negative charge to the molecule and a strong reducing agent such as
mercaptoethanol and Dithiothreitol (DTT) that could disrupt any disulfide bridge
holding protein tertiary structures. The buffer solution along with protein sample at
an appropriate pH is loaded onto the top of stacking gel with large pore size to concentrate the protein into narrow bands prior to their entry into resolving gel of
smaller pore size. Stacking gel is poured on top of the resolving gel and it is into this
gel that the wells are formed. On applying electric field, a voltage gradient is formed
between the chloride (high negative charge) and glycerine ions (low negative
charge) in the electrode buffer at pH 6.8, which serves to stack the proteins into
Recent Advances in Analysis of Food Proteins