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Nowadays, capillary electrophoresis is the most commonly used analysis technique primarily due to its faster separation times and the use of multi-capillary
arrays analysing hundreds of samples per day. Secondly, capillaries used are of
micro scale dimensions, only microlitres of reagent are needed for analysis of nanolitres of sample, along with the ability for on-line detection down to femto-mole
(10
−15
moles). The use of micro capillaries result in more heat dissipation because
of their large surface area to volume rations thus higher resolution than traditional
electrophoresis. It can be used to separate a wide spectrum of biological molecules
including amino acids, peptides, proteins, DNA fragments (e.g. synthetic oligonucleotides) and nucleic acids, as well as any number of small organic molecules such
as drugs or even metal ions. This technique was used for detection of proteolysis of
whey proteins using a coated fused-silica capillary column (Miralles et al. 2003).
CE-based separation techniques such as capillary zone electrophoresis (CZE),
Capillary gel electrophoresis (CGE), Capillary isoelectric focusing (CIEF), and
Capillary isoelectric focusing (CEC) has become a popular choice for characterization of therapeutic proteins and peptides (Creamer et al. 2014) In additional of having the potential for high throughput analysis using capillary arrays, these provide
versatile, efficient, and fast analyses of proteins.
Immuno-Precipitation (IP)
This is a technique where a protein antigen is precipitated out of a complex sample
mixture using a specific antibody coupled to sedimentable matrix. This technique
analyzes already isolated proteins by other biochemical methods such as density
gradient dependent sedmentation or gel filtration methods. The antigens isolated by
immunoprecipitation are further analysed by western blotting or SDSPAGE. Antibodies used can be either polyclonal or monoclonal obtained from various animal species. They can bind non covalently to immunoadsorbents such as
protein A or G–agarose beads, or can be coupled covalently to a solid-phase matrix
(Bonifacino et al. 2001). Immunoprecipitation or co-immunoprecipitation is used to
enrich a specific protein or protein complex from a tissue homogenate, cell lysate,
or culture supernatant (Kaboord and Perr 2008).
In IP firstly, the antigen (specific protein of interest) is enriched from tissue
homogenate, suspension cultures or cell lysates. Enrichment is achieved by binding
antigen with a specific antibody that is non-covalently attached to solid phase matrix
such as protein A or protein G aragose beads. Subsequently, enriched antigens are
incubated with immobilized antibody solid-phase matrix followed by thorough
washing. The resultant precipitated complex are denatured and resolved by SDSPAGE and can further be analysed using number of different methods such as western blot or protein mass spectrometry (Xie et al. 2017).
Recent Advances in Analysis of Food Proteins
Nowadays, capillary electrophoresis is the most commonly used analysis technique primarily due to its faster separation times and the use of multi-capillary
arrays analysing hundreds of samples per day. Secondly, capillaries used are of
micro scale dimensions, only microlitres of reagent are needed for analysis of nanolitres of sample, along with the ability for on-line detection down to femto-mole
(10
−15
moles). The use of micro capillaries result in more heat dissipation because
of their large surface area to volume rations thus higher resolution than traditional
electrophoresis. It can be used to separate a wide spectrum of biological molecules
including amino acids, peptides, proteins, DNA fragments (e.g. synthetic oligonucleotides) and nucleic acids, as well as any number of small organic molecules such
as drugs or even metal ions. This technique was used for detection of proteolysis of
whey proteins using a coated fused-silica capillary column (Miralles et al. 2003).
CE-based separation techniques such as capillary zone electrophoresis (CZE),
Capillary gel electrophoresis (CGE), Capillary isoelectric focusing (CIEF), and
Capillary isoelectric focusing (CEC) has become a popular choice for characterization of therapeutic proteins and peptides (Creamer et al. 2014) In additional of having the potential for high throughput analysis using capillary arrays, these provide
versatile, efficient, and fast analyses of proteins.
Immuno-Precipitation (IP)
This is a technique where a protein antigen is precipitated out of a complex sample
mixture using a specific antibody coupled to sedimentable matrix. This technique
analyzes already isolated proteins by other biochemical methods such as density
gradient dependent sedmentation or gel filtration methods. The antigens isolated by
immunoprecipitation are further analysed by western blotting or SDSPAGE. Antibodies used can be either polyclonal or monoclonal obtained from various animal species. They can bind non covalently to immunoadsorbents such as
protein A or G–agarose beads, or can be coupled covalently to a solid-phase matrix
(Bonifacino et al. 2001). Immunoprecipitation or co-immunoprecipitation is used to
enrich a specific protein or protein complex from a tissue homogenate, cell lysate,
or culture supernatant (Kaboord and Perr 2008).
In IP firstly, the antigen (specific protein of interest) is enriched from tissue
homogenate, suspension cultures or cell lysates. Enrichment is achieved by binding
antigen with a specific antibody that is non-covalently attached to solid phase matrix
such as protein A or protein G aragose beads. Subsequently, enriched antigens are
incubated with immobilized antibody solid-phase matrix followed by thorough
washing. The resultant precipitated complex are denatured and resolved by SDSPAGE and can further be analysed using number of different methods such as western blot or protein mass spectrometry (Xie et al. 2017).
Recent Advances in Analysis of Food Proteins