284
and secondary antibody recognises the primary antibody (recognizing one) and is
conjugated to reporter enzyme usually either horseradish peroxidase (HRP) or alkaline phosphatase that enables detection of coloured electrophoretic band indicating
position of protein of interest. In general the technique uses three elements to
accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3)
marking target protein using a proper primary and secondary antibody for visualization (Mahmood and Yang 2012).
Alkaline phosphatase converts colourless 5-bromo-4-chloro-indolylphosphate
(BCIP) substrate into a blue product and horseradish peroxidise oxidises either
3-amino-9-ethylcarbazole into an insoluble brown product, or 4-chloro-lnaphthol
into an insoluble blue product using H 2 O 2 as a substrate (Wilson and Walker 2010).
Other enzymes such as β-galactosidase, glucose oxidase and glucose- 6-phosphate
dehydrogenase can also be used. The method thus finds wide application in cell
biology and molecular biology and helps to identify specific proteins from a complex mixture of proteins extracted from cells. Due to the high resolution power of
the electrophoresis coupled with the specificity of the antibody, the method has been
used for the detection of various molecular forms of proteolytic enzymes in whey,
such as the various forms of cathepsin D; procathepsin D, pseudocathepsin D,
mature single-chained and mature two-chained cathepsin D (Larsen and Petersen
1995). It is thus very powerful for discrimination of different molecular forms of the
target protein e.g., to follow activation by proteolytic cleavage such as during
purification.
Immunoelectrophoresis
This is a two-stage technique for characterization of antibodies that combines the
principle of electrophoresis and immunodiffusion. The proteins of interest are first
separated by electrophoresis in a supporting media such as agarose and then immunodiffused with the addition of antiserum in a well that is cut from agarose gel.
When an antibody interacts with a specific antigen, a white precipitin band is formed
on black background (immunodiffusion). This precipitate band shows the presence
of antibody specific to target antigen. Presence of one precipitin band indicates
homogeneity of antibody while as antibody heterogeneity is represented by presence of multiple precipitate bands. Likewise absence of any precipitin band indicates no specific antibody against target antigen (Buyukkoroglu and Senel 2018).
Mobility of the molecules during electrophoresis is dependent on a number of factors such as net charge of molecules, size & shape of the molecules, pH of buffer &
ionic strength of buffer etc. Four types of immune-electrophoresis have been used,
such as electro-immunoassay (EIA) or rocket/Laurell rocket electro-immunoassay,
classical immune-electrophoresis, immuno-fixation electrophoresis (IFE), and capillary electrophoresis (Levinson 2009).
M. Manzoor et al.
and secondary antibody recognises the primary antibody (recognizing one) and is
conjugated to reporter enzyme usually either horseradish peroxidase (HRP) or alkaline phosphatase that enables detection of coloured electrophoretic band indicating
position of protein of interest. In general the technique uses three elements to
accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3)
marking target protein using a proper primary and secondary antibody for visualization (Mahmood and Yang 2012).
Alkaline phosphatase converts colourless 5-bromo-4-chloro-indolylphosphate
(BCIP) substrate into a blue product and horseradish peroxidise oxidises either
3-amino-9-ethylcarbazole into an insoluble brown product, or 4-chloro-lnaphthol
into an insoluble blue product using H 2 O 2 as a substrate (Wilson and Walker 2010).
Other enzymes such as β-galactosidase, glucose oxidase and glucose- 6-phosphate
dehydrogenase can also be used. The method thus finds wide application in cell
biology and molecular biology and helps to identify specific proteins from a complex mixture of proteins extracted from cells. Due to the high resolution power of
the electrophoresis coupled with the specificity of the antibody, the method has been
used for the detection of various molecular forms of proteolytic enzymes in whey,
such as the various forms of cathepsin D; procathepsin D, pseudocathepsin D,
mature single-chained and mature two-chained cathepsin D (Larsen and Petersen
1995). It is thus very powerful for discrimination of different molecular forms of the
target protein e.g., to follow activation by proteolytic cleavage such as during
purification.
Immunoelectrophoresis
This is a two-stage technique for characterization of antibodies that combines the
principle of electrophoresis and immunodiffusion. The proteins of interest are first
separated by electrophoresis in a supporting media such as agarose and then immunodiffused with the addition of antiserum in a well that is cut from agarose gel.
When an antibody interacts with a specific antigen, a white precipitin band is formed
on black background (immunodiffusion). This precipitate band shows the presence
of antibody specific to target antigen. Presence of one precipitin band indicates
homogeneity of antibody while as antibody heterogeneity is represented by presence of multiple precipitate bands. Likewise absence of any precipitin band indicates no specific antibody against target antigen (Buyukkoroglu and Senel 2018).
Mobility of the molecules during electrophoresis is dependent on a number of factors such as net charge of molecules, size & shape of the molecules, pH of buffer &
ionic strength of buffer etc. Four types of immune-electrophoresis have been used,
such as electro-immunoassay (EIA) or rocket/Laurell rocket electro-immunoassay,
classical immune-electrophoresis, immuno-fixation electrophoresis (IFE), and capillary electrophoresis (Levinson 2009).
M. Manzoor et al.