Proteins as DNA replication, RNA transcription, andProteins as DNA replication, RNA transcription, and

Proteins
are the fundamentals of biologically-active agents, such as enzymes, antibodies
and hormones. The total number of human protein products, including splice
variants and essential posttranslational modifications (PTMs), has been
estimated to be around one million.6 Proteins can not only determine
the structure of the system, the functioning and the development of living
organisms, but they also control cellular processes such as DNA replication, RNA
transcription, and protein translation.2 Gene expression carries out
all the functions essential for life; therefore, the need to separate and
analyse proteins is fundamental.

 

‘Proteomics’
is the study of proteins. This involves the use of technology to identify and
quantify proteins.4 After the success of the human genome project; proteome
is a newly emerging field of biochemistry providing the knowledge of proteins
interactions within the human body. Therefore, the study of proteomics is significant
for the development of novel drugs to control many fatal diseases.15

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The
analysis of proteins is technically challenging due to the complexity of the
samples (e.g., serum, other bodily fluids, or tissues) and the broad range of
protein concentrations.6 The number of different proteins expressed
at a given time ranges from several thousand for simple prokaryotic organisms
and up to a 10,000 in eukaryotic cell extracts.5 Current proteomic
studies have shown that the majority of proteins involved in gene expression
are more easily identified, they are found to be present in numbers of 105-106
molecules per cell, whereas receptor proteins are present in much lower
concentrations of 100 molecules per cell, these are usually not detected.5
This highlights the importance for the requirement for an improved method which
can identify low-abundance proteins.2 Due to the complexity of the
tested material, the use of high-resolution, high selectivity and sensitivity
separation techniques are of prime importance. Efficient sample preparation to
account for lower abundance components, extensive data processing and data
analysis are also required.6

 

To
study the structure of a protein it must first be purified. However, because
proteins vary in size, charge, and water solubility, one single method cannot be
used to isolate all proteins. The process requires methods both for separating
proteins and for detecting the presence of specific proteins.9
Protein fractionation techniques can be divided into two main categories:
gel-based and gel-free. For example, all types of one- and two-dimensional
electrophoresis, such as SDS-PAGE, native PAGE, and isoelectric focusing using
immobilised pH gradient gel strips, are gel-based.10 Two-dimensional
polyacrylamide gel electrophoresis (2-DE) is currently the most widespread
technique used in the study of proteomes. 2-DE is a form of gel electrophoresis
used to analyse individual proteins from complex samples. Since the first
publication of 2-DE in 1975 by O’Farrell and Klose, the use of 2-DE has
attracted growing interest.2 Coupling 2-DE with immobilised pH
gradients, IPG-Dalt (2DGE), has provided exceptionally high resolution,
improved reproducibility, and high loading capacity rendering the method as fit
for its purpose. 2DGE is capable of separating over thousand different proteins
in one gel.7