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2D DIGE is a scientific technique that is a modified version of 2DE or two dimensional electrophoresis. It helps to differentiate two to three protein samples simultaneously, on the same gel and here the proteins are covalently bonded with fluorescent dyes for identification. All the protein samples are tagged with Cy3, Cy5, as well as Cy2 fluorophore and this technique is beneficial to detect even 0.5fmol of protein. This is N-hydroxysuccinimidyl (NHS) ester derivatives of cyanine dyes. 2D DIGE is one of common and well-established methods, which played a significant role in proteomic analysis (Zhan et al. 2019). 2D DIGE is a common method, which helps to resolve huge numbers of protein separation in a single-run. In traditional 2-DE there were a number of limitations and in order to overcome this intrinsic gel-to-gel variability, this 2D DIGE technique emerged. One can expect very sensitive protein separation as well as linear detection in an extensive range of protein structure. Guerini et al. (2020) stated that to determine longevity of aged women the two differential gel-electrophoresis is one of common and most significant method.
Above figure denotes that label of protein sample with different CyDyes, which combine into a single tube. In next stage, two dimensional differences gel electrophoresis performed and in next stage differentiated images based on Cy2, Cy3 and Cy5 (Guest, 2017).
Cy3 and Cy5 are used to tag the control proteins and treatment proteins in one sample and vice versa in another sample. These two dyes are matched according to the size and charge according to the protein samples to ensure co-migration in the gel (Meleady, 2018). However, Cy2 remains constant for doing all the internal standards. Minimum labelling with lysine labelling is done and the reason for using lysine labelling is that in almost all protein residues contain at least one lysine residue in its structure. Therefore, the targeted protein, having lysine in its structure will get easily detected by this lysine labelling. In other cases, saturation labelling of cysteine can be used if the targeted protein does not contain lysine in its structure (Pasquali et al. 2017) The CyDyes react with the primary amine group of the targeted proteins by a reaction called nucleophilic substitution. At First the lysis buffer is prepared having pH of 8.5 with no IPG buffers or carrier ampholytes. Only 50 microgram protein is labelled with 400 moles of dye on ice-water for around 30 minutes. After that pooled internal standards are prepared by combining equal amounts of proteins from control and treatment samples and are dyed with Cy2 dye. This internal standard has various advantages such as every sample of protein appears on gel, reduces gel-to-gel variation, and a reference point is achieved. After the internal standard and treatment and protein samples are labelled they are mixed together, the mixture of samples is then rehydrated and can be focused on an isoelectric focusing instrument (IEF). After the first and second equilibrium steps it is run on SDS-PAGE and all the protein samples from control or treatment get separated in one single gel and scanned by fluorescent scanner and Cy2, Cy3, Cy5 patterns can be obtained. Thus 2D DIGE technique has resolved the previous 2DE issues regarding protein sample separation and identification.
This section is going to draw a comparison among two dimensional differences gel electrophoresis with two dimensional gel electrophoresis. It has to be mentioned here that the alternative method chosen here is two dimensional gel electrophoresis.
This section is going to focus on application of two-dimensionaldifference gel electrophoresis for the medical aspect. In context of application, this study is only focus on cancer disease and application of two-dimensional differences gel electrophoresis. Cancer is a condition that results from the genetic mutation due to environmental or hereditary factors. The DNA that transcribed to form RNA and RNA that translates to produce protein. 2D DIGE is a useful technique to study the complex interaction of the protein and the biological activities in the body. The reason why it is called a type of proteomics that gives highly accurate and reliable result to identify proteins in control and treatment samples. Therefore, it can be used to detect cancer biomarkers in the body. Study says, 2D DIGE is useful in detection of gastric cancer, colorectal cancer, liver cancer, lung cancer, oesophageal cancer. Here, the control protein sample contains heathy proteins and the treatment sample contains protein from the diseased patient (Kondo, 2019). A two-dimensional differential gel electrophoresis process is undertaken to identify protein, which is responsible for the cancer. Therefore, by focusing on the above discussion, it can be stated that two-dimensional gel electrophoresis has a significant role in the medical aspect.
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Guerini FR, Torretta E, Trecate F, Cesari M, Mari D, Clerici M, &Gelfi C. (2020). Can Serum Nitrosoproteome Predict Longevity of Aged Women? Int J Mol Sci. 2020 Nov 27;21(23):9009. doi: 10.3390/ijms21239009. PMID: 33260845; PMCID: PMC7731247.
Guest, P. C. (2017). A Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) Protocol for Studies of Neural Precursor Cells. In Proteomic Methods in Neuropsychiatric Research (pp. 183-191). Springer, Cham.
Kondo, T., 2019. Cancer biomarker development and two-dimensional difference gel electrophoresis (2D-DIGE). BiochimicaetBiophysicaActa (BBA)-Proteins and Proteomics, 1867(1), pp.2-8. https://sci-hub.hkvisa.net/10.1016/j.bbapap.2018.07.002
Meleady, P., 2018. Two-dimensional gel electrophoresis and 2D-DIGE. Difference Gel Electrophoresis, pp.3-14.https://link.springer.com/protocol/10.1007/978-1-4939-7268-5_1
Pasquali, M., Serchi, T., Planchon, S. and Renaut, J., 2017. 2D-DIGE in proteomics. In Functional Genomics (pp. 245-254). Humana Press, New York, NY. https://link.springer.com/protocol/10.1007/978-1-4939-7231-9_17
Zhan, X., Li, B., Zhan, X., Schlüter, H., Jungblut, P. R., &Coorssen, J. R. (2019). Innovating the concept and practice of two-dimensional gel electrophoresis in the analysis of proteomes at the proteoform level. Proteomes, 7(4), 36.https://www.mdpi.com/2227-7382/7/4/36/pdf
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