288
narrow bands between the ions. Migration into the resolving gel of a different pH
8.8 disrupts this voltage gradient and allows separation of the proteins into discrete
bands (Nielsen 2010).
The sample buffer also contains an ionisable tracking dye, (Bromophenol blue)
that monitors electrophoretic separation progress, and sucrose or glycerine, which
gives the sample solution density allowing the sample to settle easily at the bottom
when injected into the loading well. Being a small molecule, the dye migrates ahead
of proteins and remains totally un-retarded till it reaches the bottom of gel where
current is turned off. On completion of electrophoresis protein band are made visible as blue bands on clear background by treating gel with protein dye (Coomassie
Brilliant Blue) followed by de-staining to removes unbound background dye from
the gel. Specific antibodies or enzyme stains can be used to detect a protein of
interest.
Two-Dimensional Gel Electrophoresis (2-DE)
2-DE technique was initially introduced in 1975 by P. H. O’Farrell and J. Klose
(O’Farrell 1975; Klose 1975). It is one of the leading powers in proteomics and
study of proteins. This is the most versatile method for fractionating and visualizing
advanced protein complex extracted from cells, tissues, or alternative biological
samples with an exceptional ability to separate thousands of proteins at once. It is
the only currently available method which is capable of separating thousands of
protein molecules by two consecutive techniques. In the first step, isoelectric focusing separates proteins according to their charge and in second step; proteins are
separated according to their molecular mass in a single gel. The separated protein
on the gel with isoelectric focusing is negatively charged by treatment with SDS,
and the electrophoresis is performed by inserting the gel horizontally into the SDSPAGE gel. The separated compounds are visualized by staining with Coomassie
stains, silver stains or fluorescent dyes. Following separation by 2-DE, the protein
spot detected on gel are cut out, de-stained, digested for further analyses of typtic
peptides by peptide fingerprinting using MALDI-TOF or nano-LC-ion trap mass
spectrometry or probed by antibodies, then followed by computer- assisted software
for image evaluation. This technique finds wide application in detection of post- and
co-translational protein modifications, study cell differentiation(Jungblut and
Seifert 1990), detection of biomarkers and disease markers, drug discovery, cancer
research(Wu et al. 2002), bacterial pathogenesis (Enany et al. 2013), purity checks,
micro scale protein purification, and product characterization.
Although 2 DE is capable of high resolution, it has some limitations, including
limited molecular mass range, poor separation of highly acidic or basic proteins,
and exclusion of the majority of membrane proteins (Kline and Wu 2009). Modern
proteomics approaches combine high resolution 2-DE technique with mass spectrometry using soft ionisation such as matrix-assisted laser desorption/ionisation
(MALDI) or electrospray ionisation (ESI) followed by time-of-flight (TOF), ion
M. Manzoor et al.
narrow bands between the ions. Migration into the resolving gel of a different pH
8.8 disrupts this voltage gradient and allows separation of the proteins into discrete
bands (Nielsen 2010).
The sample buffer also contains an ionisable tracking dye, (Bromophenol blue)
that monitors electrophoretic separation progress, and sucrose or glycerine, which
gives the sample solution density allowing the sample to settle easily at the bottom
when injected into the loading well. Being a small molecule, the dye migrates ahead
of proteins and remains totally un-retarded till it reaches the bottom of gel where
current is turned off. On completion of electrophoresis protein band are made visible as blue bands on clear background by treating gel with protein dye (Coomassie
Brilliant Blue) followed by de-staining to removes unbound background dye from
the gel. Specific antibodies or enzyme stains can be used to detect a protein of
interest.
Two-Dimensional Gel Electrophoresis (2-DE)
2-DE technique was initially introduced in 1975 by P. H. O’Farrell and J. Klose
(O’Farrell 1975; Klose 1975). It is one of the leading powers in proteomics and
study of proteins. This is the most versatile method for fractionating and visualizing
advanced protein complex extracted from cells, tissues, or alternative biological
samples with an exceptional ability to separate thousands of proteins at once. It is
the only currently available method which is capable of separating thousands of
protein molecules by two consecutive techniques. In the first step, isoelectric focusing separates proteins according to their charge and in second step; proteins are
separated according to their molecular mass in a single gel. The separated protein
on the gel with isoelectric focusing is negatively charged by treatment with SDS,
and the electrophoresis is performed by inserting the gel horizontally into the SDSPAGE gel. The separated compounds are visualized by staining with Coomassie
stains, silver stains or fluorescent dyes. Following separation by 2-DE, the protein
spot detected on gel are cut out, de-stained, digested for further analyses of typtic
peptides by peptide fingerprinting using MALDI-TOF or nano-LC-ion trap mass
spectrometry or probed by antibodies, then followed by computer- assisted software
for image evaluation. This technique finds wide application in detection of post- and
co-translational protein modifications, study cell differentiation(Jungblut and
Seifert 1990), detection of biomarkers and disease markers, drug discovery, cancer
research(Wu et al. 2002), bacterial pathogenesis (Enany et al. 2013), purity checks,
micro scale protein purification, and product characterization.
Although 2 DE is capable of high resolution, it has some limitations, including
limited molecular mass range, poor separation of highly acidic or basic proteins,
and exclusion of the majority of membrane proteins (Kline and Wu 2009). Modern
proteomics approaches combine high resolution 2-DE technique with mass spectrometry using soft ionisation such as matrix-assisted laser desorption/ionisation
(MALDI) or electrospray ionisation (ESI) followed by time-of-flight (TOF), ion
M. Manzoor et al.