Environmental Engineering Reference
In-Depth Information
Fig. 15.3 Graphic illustration 2D PAGE. Panel (a) shows Iso electric focussing of the proteins
based on the pI. Panel (b) illustrates SDS-PAGE wherein the proteins are separated based on their
molecular weight
15.1.7 Advantages and Limitations of Method
The main advantage of using 2D-PAGE/DIGE is the large mass range and the large
amount of proteins that can be analysed in a single run. 2D-PAGE/DIGE
are particularly good for proteins within the mass range of 20-250 kDa and pI of
3-11. Proteins of particular pI and mass can be focused on by using narrow range
pH isoelectric focusing strips and the percentage of acrylamide used in the second
dimension PAGE gel, respectively. If sufficient sample is present on the gel
(
>
300
μ
g of total protein) then proteins can be excised from the gel, subjected to
in-gel proteolysis and analysed by mass spectrometry.
The 2-D SDS-PAGE and 2D-DIGE approaches to protein profiling are accessi-
ble and economical methods that possess high resolving power. The problem of
reproducibility and quantisation has been greatly improved with the use of
2D-DIGE. IPGs have enhanced the resolution which enables the analyst to tailor
the pH gradient for maximum resolution using ultra zoom gels with a narrow pH
gradient range. With modern 2D-PAGE, it is also possible to resolve two proteins
that differ in pI by 0.001 U. The use of imagers and computers allows fast data
mining, acquisition, analysis, spot detection, normalisation, protein profiling, back-
ground correction and reporting and exporting of data.
Along with several advantages, 2-D PAGE also involves a few limitations.
2D-PAGE/DIGE is a lower throughput and time-consuming process (3-4 days
per run) that involves several steps and requires high practical skills. 2D-PAGE/
DIGE is not a good technique for the analysis of extremely acidic, basic or
