How does 2-D gel electrophoresis work? One-dimensional protein gel electrophoresis techniques such as SDS-PAGE are routinely performed in the lab, these methods offer low separation capability. Two-dimensional gel electrophoresis (2-DE) separates proteins in complex samples by pI value and molecular weight, enabling the direct comparison of hundreds or thousands of proteins simultaneously with high resolution. When paired with analytical software, immunodetection, or mass spectrometry techniques, 2-DE provides a powerful tool that aids in protein identification and other proteomic analyses. For additional resources and products, please visit our Protein Electrophoresis and Western Blotting page.
Two-dimensional gel electrophoresis has generally been regarded as difficult to perform and time-consuming, requiring advanced user training while offering low reproducibility and high inter-operator variability. The Auto2D® system fully automates two-dimensional gel electrophoresis. The applied sample enters the first-dimension gel passively, separating proteins by their isoelectric points. After equilibration, the first-dimension gel is set onto a horizontal SDS-PAGE gel, which resolves proteins by molecular weight. The Auto2D® 2-D Electrophoresis Device is user-friendly and enables two-dimensional electrophoresis in 1-2 hours for faster, more reproducible results. Additional features and benefits of the Auto2D® 2-D Electrophoresis Device include:
The Auto2D® 2-D Electrophoresis Device fully automates two-dimensional gel electrophoresis, simplifying protein analysis and providing more consistent, reproducible results that are user-independent. The efficient engineering of the Auto2D® system significantly reduces the amount of time spent during sample loading, isoelectric focusing, equilibration, and SDS-PAGE from 4-24 hours to only 1-2 hours. This makes the Auto2D® device unique compared to other semi-automated 2-DE systems on the market.
Figure 1.Two-dimensional gel electrophoresis workflow and the steps that are automated by the Auto2D® device.
The Auto2D® 2-D Electrophoresis Device has been proven in numerous applications. Examples of suitable use cases include differential protein expression analysis in cancer research and the elucidation of disease mechanisms. Additional applications include the use for separating purified proteins for crystallization or post-translational modification analysis and 2-D western blotting in research areas such as allergy research or cell signaling pathway analysis.
Figure 2.Left: Differential protein expression analysis in cultured cells for cancer biomarker research. Green represents more abundant proteins in normal cells. Red represents cancer-specific protein spots. Yellow represents the overlap in protein expression. Right: Differential protein expression in plasma samples.
Figure 3.Left: Assessing microheterogeneities in therapeutic antibody drug products. Red triangle indicates spots for target antibody; lower spots are antibody missing regions. Right: Protein analysis before and after purification. 2-DE can be used to check whether the sample is homogeneous for crystallization and X-ray structural analysis.
Figure 4.Flour extract proteins (detected by SYPRO Ruby) by SDS-PAGE (A), WB: Anti-Gliadin antibody by SDS-PAGE (B), and merge by SDS-PAGE (C). Flour extract proteins (detected by SYPRO Ruby), arrows indicate spots detected by Anti-Gliadin antibody (D). WB using Anti-Gliadin antibody (E), and merge (F).
2-D gel electrophoresis is also suitable for contaminating host cell protein (HCP) analysis in therapeutic protein and antibody bioprocessing. Residual HCP impurities introduced during biologics manufacturing processes can trigger immunogenic responses in patients and reduce drug efficacy. Regulatory compliance calls for the monitoring and removal of HCPs to acceptably low levels to ensure safety. Pharmacopoeias in Japan, Europe, and the United States have published guidelines for the development and validation of HCP assays for biopharmaceutical product manufacturing.
To meet compliance, HCP assays must be methodically developed with rigorous qualification of all assay components. For antibody validation, two dimensional polyacrylamide gel electrophoresis (2D-PAGE) is required to ensure comprehensive HCP coverage. Gel electrophoresis methods including 2D-PAGE are also advised for the characterization of antigens used as calibration standards or immunogens for polyclonal antibody production. The Auto2D® 2-D Electrophoresis Device eliminates the need for advanced user training, facilitating implementation into bioprocess workflows to meet compliance with industry standards.
ELISAs are the most common assay format for HCP analysis. This immunoassay method relies on polyclonal antibodies that recognize residual HCPs with broad range coverage. Japanese and European Pharmacopoeia specify the use of 2-D gel electrophoresis followed by Western blot analysis (2-D Western blotting) to evaluate HCP antibody coverage. U.S. Pharmacopoeia specifies the use of 2-D Western blotting or immunoaffinity purification followed by gel electrophoresis to evaluate antibody coverage.
Figure 5.20 μg of Cy3-labeled CHO HCP antigen from a commercial source was separated by 2-D gel electrophoresis on the Auto2D® system using a pH 3-10 IEF chip and 12.5% PAGE chip (A). Proteins were then transferred to membranes and analyzed by Western blotting using two different anti-HCP antibodies (B, C). Data indicate that anti-HCP antibody 1 has broader coverage of host cell proteins, compared to anti-HCP antibody 2.
Consistent, reproducible processes are essential in biologics manufacturing and quality control. In addition to faster results and higher throughput, the fully automated Auto2D® gel electrophoresis system eliminates day-to-day and inter-operator variability in 2D-PAGE and 2D-DIGE methods, offering significantly better reproducibility for more reliable results.
Figure 6.0.75 μg of Cy5-labeled CHO HCP antigen from a commercial source was separated by 2D gel electrophoresis and analyzed on the Auto2D® system using a IEF Chip pH 3-10NL and PAGE Chip 12.5%. The same sample was measured a total of three times (A-C). White spots (D) show overlap of results. The data suggest high reproducibility of two-dimensional gel electrophoresis using the Auto2D® system.
Immunization with HCP antigen is required to obtain polyclonal antibody pools for ELISA and other immunoassays. HCP antigen is also used as a calibration standard for detection and quantitation. Process-specific HCP antigens are generated using non-transfected null cell cultures. These antigens must be characterized to show the pattern of HCPs, confirm the presence of a broad spectrum of proteins, and demonstrate that HCP antigens from null cultures are representative of those in production cultures. Japanese, European, and U.S. Pharmacopoeia advise the use of SDS-PAGE or 2-D gel electrophoresis for antigen characterization and profiling. U.S. Pharmacopoeia also suggests the use of 2-D gel electrophoresis as a complementary method for residual HCP monitoring.
Figure 7.A mixture of Cy3-labeled unpurified CHO HCP antigen and Cy5-labeled Protein A-purified CHO HCP antigen were separated and analyzed by two-dimensional difference gel electrophoresis (2D-DIGE) using the Auto2D® system (A-B). Red spots in the merged data (C) represent host cell proteins which tend to remain even after Protein A purification. These data demonstrate the use of the Auto2D® system for profiling remaining host cell protein components post-purification.
The Auto2D® system helps you meet compliance with regulatory requirements for HCP analysis in biopharmaceutical manufacturing. This fully automated system eliminates day-to-day and inter-operator variability for reliable results in less than 2 hours. Easy to implement into bioprocess workflows, the Auto2D® system reduces lab downtime associated with production changes. There’s no need to outsource – protect your intellectual property using in-house resources for HCP analysis. For additional products and resources, please visit our Protein Electrophoresis and Western Blotting hub page.