DIGE 2D Gel Electrophoresis
DIGE 2D gel electrophoresis is a classic protein separation technique that plays a crucial role in proteomics research. Utilizing two orthogonal separation mechanisms-Isoelectric Focusing (IEF) and SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)-this method effectively separates proteins on a two-dimensional plane. This allows for the clear analysis of thousands of proteins in complex samples. Since its inception in the 1970s, it remains an essential tool in proteomics due to its high resolution and the ability to provide intuitive insights into protein expression patterns. In life sciences, it finds broad applications in disease biomarker discovery, drug target identification, environmental toxicology, and food science. For instance, in cancer research, this technique has been employed to investigate proteomic alterations in types of cancer such as breast, gastric, and liver cancer, offering new perspectives for early diagnosis and targeted therapy. In the study of neurodegenerative disorders like Alzheimer's disease, it aids in exploring critical molecular events such as neuronal apoptosis and synaptic protein abnormalities. Furthermore, in the field of environmental science, this method is utilized to assess the effects of environmental pollutants like heavy metals and pesticides on protein expression in organisms, thereby evaluating potential ecological risks. In food science and agricultural research, it helps in examining protein changes during food processing, such as dairy fermentation and grain milling, to optimize food quality. Additionally, it assists in studying plant protein expression under various environmental conditions, shedding light on crop resistance mechanisms, which supports agricultural breeding and crop enhancement efforts.
The principle of DIGE 2D gel electrophoresis involves a two-step separation process. The first step, isoelectric focusing (IEF), allows protein samples to migrate in a gel strip with a pH gradient (IPG strip) until they reach their isoelectric points (pI), where the net charge is zero, thus stopping their movement. This results in the formation of distinct focused bands on the IPG strip based on differing isoelectric points. Following the IEF, the IPG strip is transferred to an SDS-PAGE gel for the second dimension separation, which sorts proteins by molecular weight. SDS, as a strong anionic detergent, ensures that all proteins attain a uniform charge density, allowing them to migrate and separate solely based on molecular weight. Afterward, proteins are visualized on the gel as individual spots through staining methods like Coomassie Brilliant Blue, silver, or fluorescent staining. Researchers can then observe variations in protein expression and proceed to protein identification using mass spectrometry.
DIGE 2D gel electrophoresis boasts high resolution and visualization capabilities, enabling researchers to discern comprehensive protein expression changes, which is particularly beneficial for differential expression analysis under varying experimental conditions. The technique is relatively cost-effective when compared to some advanced proteomics analysis methods, making it the preferred choice for many laboratories engaged in proteomics research. However, it has limitations, such as reduced separation efficiency for high molecular weight proteins (>150 kDa) and low molecular weight proteins (<10 kDa). It also faces challenges in effectively detecting strongly hydrophobic membrane proteins due to solubility issues. Additionally, the dynamic range of this method is relatively narrow, leading to insufficient sensitivity for very low abundance proteins, which may result in some key regulatory proteins being overlooked. Despite these limitations, with ongoing advancements in gel electrophoresis technology and its integration with advanced mass spectrometry and bioinformatics tools, this technique continues to be valuable in research.
MtoZ Biolabs, leveraging years of expertise in proteomics research, offers high-quality DIGE 2D gel electrophoresis services for researchers. We have optimized processes for sample preparation, gel separation, protein staining, and data analysis to ensure that experimental data are highly repeatable and reliable.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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