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Proteomics, a field dedicated to studying proteins on a grand scale, has played a pivotal role in revolutionizing our comprehension of cellular processes and functions. It encompasses a diverse array of techniques aimed at characterizing proteins, including their abundance, post translational modifications (PTMs), and interactions. Among the powerful tools in the realm of proteomics, 2D electrophoresis firmly stands out as an esteemed technology for not only separating and analyzing intact proteins from complex mixtures, but also pushing the boundaries of innovation.
Sometimes referred to as 2-DE, two-dimensional gel electrophoresis serves as a potent and widely renowned method for scrutinizing protein mixtures through the use of gel electrophoresis. Although isoelectric focusing and SDS-PAGE have proven their effectiveness as individual strategies, 2D electrophoresis ingeniously combines the strengths of both techniques. By employing isoelectric focusing initially, proteins are separated and their isoelectric point (pl) is determined. Subsequently, SDS-PAGE is employed to separate these proteins once again, facilitating the identification of their molecular weight. Harnessing the power of high-throughput mass spectrometry, each protein spot on a 2D electrophoresis can be eluted and accurately identified.
As a result, 2D electrophoresis plays an instrumental role in facilitating comparisons of protein profiles between tissues, circumstances, or between samples that remain untreated and those that have undergone treatment. Its contribution resonates, thereby forging a path towards expanded knowledge and understanding of proteins in various contexts.
Our 2D Electrophoresis Service
The comprehensive 2D electrophoresis service provided by Creative Proteomics, a leading company in the field of proteomics, continuously optimizes service workflow and utilizes the advantages of 2D-DE technology and mass spectrometry to assist scientists in better protein analysis and PTM research. With this service, complex mixtures consisting of thousands of different proteins can be separated and their relative amount can then be determined.
The service offers extreme high-resolution 2D gel electrophoresis, allowing for the separation of various protein-containing samples. With different gel sizes available, ranging from 8x7 cm to 60x30 cm, the service can accommodate diverse sample characteristics and achieve exceptional resolution. Depending on the gel size and sample properties, up to an impressive 10,000 protein spots can be resolved, providing unparalleled insights into the proteome.
Advantages of Our 2D Electrophoresis Service
High-throughput: 2D electrophoresis can accurately analyze thousands of proteins in a single run.
High resolution. This technology resolves proteins according to both pI and molecular mass, and enables the characterization of proteins with posttranslational modifications that affect their charge state.
Various computer-based tools are available: We have tools such as SameSpots, Delta2D, ImageMaster, which can be used for detection and quantification of protein spots.
Cost-efficient and affordable: While mass spectrometers represent a significant investment and require experience staff, 2D electrophoresis is relatively inexpensive.
High Flexibility: We work closely with you to design the optimal experimental scheme to meet your research objectives on budget.
Protein Profiling Service by 2D Electrophoresis
Efficiently and accurately identify proteins in SDS-PAGE and 2-DE samples are critical for analyzing proteins in complex mixtures. Two-dimensional electrophoresis can separate thousands of protein spots in a single separation, making it ideal for protein profiling studies.
By comparing 2D gel images from different samples or conditions, researchers can detect differences in protein expression levels, indicating the presence or absence of specific proteins, changes in protein abundance, or the occurrence of posttranslational modifications (PTMs).
Protein profiling is a powerful tool for biomarker discovery, where differences in protein expression can be used as diagnostic or prognostic markers for diseases.
Protein profiling can be used to describe the distribution of protein isoforms and species, which can reveal details about alternative splicing and protein variants.
It enables an examination into protein quantification, which entails figuring out how much protein abundance has changed between samples or experimental groups.
Protein profiling can also be used to measure host cell protein (HCP) antibody coverage and find HCP contaminants, guaranteeing quality control in the development of biopharmaceuticals.
PTM Studies by 2D Electrophoresis
Because of its capacity to differentiate charge and size isomers of polypeptides, 2D electrophoresis is useful for researching posttranslational modifications (PTMs).
Profiling of phosphoprotein expression: Phosphorylated proteins migrate to a more acidic part of the gel, allowing researchers to investigate signaling pathways and identify phosphorylation events.
Acetylated protein expression profiling: acetylation of proteins at their amino termini or on lysine side-chains will cause them to shift to a more acidic region on the gel. Acetylation of proteins can affect protein function, interactions and subsequent or additional posttranslational modifications
Methylated protein expression profiling: Methylation alters the isoelectric point of proteins and has implications in epigenetic studies and gene regulation.
Glycosylated protein expression profiling: Glycosylation, a complex PTM, can result in changes in molecular weight and isoelectric point, making 2D electrophoresis suitable for its analysis.
2D DIGE Images for various Sample Types
|Human Cells||Human Fluid/Tissue||Lab Animals||Bacteria & Others|
|Jurkat cells||Breast cancer cell line||Saliva||Mouse & Rat brain||E. coli|
|HEK293||Hepatocytes||Amniotic fluid||Mouse retina cells||Bacteria-secreted|
|Blood T cells||Keratinocytes||Adipose||Rat tear||Yeast|
|Blood monocyte||Platelets||Liver tissue||Drosophila||Plant|
|Dendritic cells||Blood cell exosome||Serum / Plasma||C. elegans||Rice|
|Cerebrospinal fluid||Zebrafish||Flu vaccines|
Advantages of 2D DIGE Technology
A notable advancement in 2D electrophoresis is the introduction of 2D DIGE (Difference Gel Electrophoresis), which offers significant advantages over standard 2D gel electrophoresis and other proteomics techniques. One of the key advantages is its higher sensitivity. 2D DIGE employs fluorescent labeling with a sensitivity of 0.2 ng/spot, surpassing the sensitivity of standard 2D gel electrophoresis with Coomassie blue staining (100 ng/spot) or silver staining (1 ng/spot). This heightened sensitivity enables the detection of subtle changes in protein abundance, as small as 10%.
Another remarkable advantage of 2D DIGE is its higher accuracy. The technology allows for extremely high spot resolution, enabling precise spot quantitation. Even differences in protein expression as small as 10% can be reliably detected, facilitating the identification of differentially expressed proteins and providing valuable insights into biological processes.
Moreover, 2D DIGE offers exceptional reproducibility. Nearly identical data can be obtained from the same sample labeled with different fluorescent dyes on the same gel or across different gels, eliminating the need for running technical replicates. This robust reproducibility enhances the reliability of the results, ensuring consistent and trustworthy analyses.
Furthermore, 2D DIGE provides a broader spectrum of protein detection. With large 2D gel formats capable of resolving approximately 5000 protein spots, the technique enables the detection and quantitation of low-abundance proteins, large proteins, and small peptides. This wide dynamic range expands the scope of proteome analysis, facilitating the comprehensive exploration of protein profiles and posttranslational modifications.
2D Electrophoresis Workflow
The 2D electrophoresis service at Creative Proteomics follows a well-established workflow, meticulously designed to deliver high-quality results. The workflow involves several key steps:
Sample Preparation: Samples, such as purified proteins, frozen cell pellets or tissues, body fluids, cell cultures, microorganisms, plants, and more, undergo customized sample preparation protocols. This step ensures the conversion of samples to a physicochemical state suitable for 2D electrophoresis.
First-Dimension Separation by Isoelectric Focusing (IEF): In the first dimension, proteins are separated based on their isoelectric point (pI). A protein mixture is loaded at the basic end of the pH gradient gel, and under the influence of an electric field, proteins with a positive net charge migrate towards the cathode, while those with a negative net charge migrate towards the anode.
Second-Dimension Separation by SDS-PAGE: After the first-dimensional separation, the proteins are immobilized on the gel strip. The gel strip is then placed on top of an SDS-PAGE gel for the second-dimensional separation based on molecular weight. This step further resolves the proteins, allowing for precise profiling and analysis.
Visualization of Results: The separated proteins can be visualized using various staining techniques, such as blue staining or silver staining. These visualization methods enable the direct observation of protein profiles and facilitate the identification of differentially expressed proteins or PTMs.
Further Analysis: Following visualization, the protein spots of interest can be eluted and subjected to downstream analyses. Information such as molecular weight, pI, and abundance can be determined. High-throughput mass spectrometry is commonly employed for protein identification, enabling comprehensive characterization of the proteome.
Figure 1. The workflow of 2D electrophoresis.
1. Why is 2D electrophoresis the benchmark technology for protein analysis?
2D electrophoresis offers the ability to separate intact proteins based on their pI and molecular weight, enabling comprehensive protein profiling and analysis.
2. What are the advantages of 2D DIGE over standard 2D gel electrophoresis?
2D DIGE provides higher sensitivity, accuracy, and reproducibility. It offers a broader spectrum of protein detection and requires fewer gels, making it a cost-efficient choice.
3. How does 2D electrophoresis contribute to biomarker discovery?
By comparing protein profiles between normal and disease states or different experimental conditions, 2D electrophoresis allows for the identification of differentially expressed proteins that may serve as potential biomarkers.
4. Can 2D electrophoresis detect posttranslational modifications (PTMs)?
Yes, 2D electrophoresis is well-suited for PTM studies. It can detect PTMs such as phosphorylation, acetylation, methylation, glycosylation, and others, providing insights into protein regulatory mechanisms.
5. What types of sample preparation are required for 2D electrophoresis?
Sample preparation protocols vary depending on the sample type. Creative Proteomics offers customized sample preparation to ensure optimal results.
6. How long does a typical 2D electrophoresis experiment take?
The timeline for a 2D electrophoresis experiment can vary based on project requirements and sample complexity. Creative Proteomics strives for fast turnaround times, typically providing results within 5-7 days.
7. Can Creative Proteomics provide additional data analysis and interpretation services?
Yes, Creative Proteomics offers comprehensive data analysis services, including protein identification through mass spectrometry, spot picking, cluster analysis, and validation through techniques such as 2D Western blotting.