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Two dimensional gel electrophoresis (2-DE)
The document describes two-dimensional gel electrophoresis (2-DE) as a powerful proteomics tool for separating complex protein mixtures through isoelectric focusing (IEF) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It details the processes involved in both dimensions, including sample preparation and visualization techniques like silver and Coomassie blue staining. Additionally, it highlights the limitations of manual comparisons in large studies and mentions available software for gel analysis, along with services offered by Creative Proteomics for protein identification.
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Two dimensional gel electrophoresis (2-DE)
- 1. Two dimensional gel electrophoresis(2-DE) Presented by Creative Proteomics
- 2. Introduction Two-dimensional gel electrophoresisis considered a powerful tool for proteomics work. It is used for separation and fractionation of complex protein mixtures from biological samples 01 The first one is called isoelectric focusing (IEF) which separates proteins according to isoelectric points (pI) 03 Thus, thousands of proteins can be separated, and the information about IEF and molecular weights can be obtained 02 The second step is SDS-polyacrylamide gel electrophoresis (SDS- PAGE) which separates proteins based on the molecular weights
- 3. The Process of2-DE Visualization of results: Staining Second dimension: SDS-PAGESample preparation First dimension: Isoelectric Focusing (IEF) 1 2 3 4 • The native samples need to be converted to a physicochemical state suitable for the first dimension IEF and keep the native charge and Mr • The sample preparation is various • The process will result in the complete solubilization, disaggregation, denaturation, and reduction of the proteins
- 4. First dimension: IsoelectricFocusing (IEF) Proteins are amphoteric molecules and the positive, negative, or zero net charge they carry depending on the pH of the surroundings. The isoelectric point (pI) is defined as the pH of a solution at which the net charge of the protein becomes zero. A protein with a positive net charge will migrate toward the cathode, becoming less positively charged until reaching its pI. While a protein with a negative net charge will migrate toward the anode, becoming less negatively charged until it also reaches its pI.
- 5. First dimension: IsoelectricFocusing (IEF) Low pH High pH Mixture of Proteins Apply electric field + - Smaller, more acidic proteins Larger, more basic proteins + - More acidic proteins More basic proteins • A protein mixture is loaded at the basic end of the pH gradient gel. • After applying an electric field, the proteins are separated depending on charges, focusing at positions where the pl value is equivalent to the surrounding pH. • Larger proteins will move more slowly through the gel, but with sufficient time will catch up with small proteins of equal charge.
- 6. Second dimension: SDS-PAGE Beperformed on flatbed or vertical systems on a slab gel The second dimension is often performed by SDS-PAGE (SDS-polyacrylamide gel electrophoresis), which is an electrophoretic method for separating polypeptides according to their molecular weights (Mr) This method often contains four steps, including preparation of the gel, the equilibrium of the IPG strips in SDS buffer, placing the equilibrated IPG strip on the SDS gel, and finally handling the electrophoresis
- 7. Second dimension: SDS-PAGE + - Moreacidic proteins More basic proteins • SDS can make proteins denaturing and bind to the backbone at a constant molar ratio. • When applying SDS and a reducing agent (like a DTT which can cleave disulfide bonds), proteins unfold into linear chains with negative charge proportional to the polypeptide chain length. • Polyacrylamide forms a mesh-like matrix which is appropriate for separating proteins. • When proteins are separated by SDS-PAGE, smaller proteins migrate faster since the less resistance. - +
- 8. Visualization of results:Staining • Sensitive and non-radioactive method • The amino acid side chains can bind to silver ions, primary the sulfhydryl and carboxyl groups of proteins, followed by reduction to free metallic silver • The protein bands are visualized as spots where the reduction occurs • Silver staining is suitable for low protein levels because of its sensitivity (in the very low ng range) Silver staining
- 9. Visualization of results:Staining Coomassie Blue staining • A relatively simple method and more quantitative than silver staining • It is suitable to detect protein bands containing about 0.2 μg or more proteins. • The Coomassie dye binds to proteins to form a protein-dye complex through Van der Waals attractions • There are two kinds of Coomassie dyes, R250 and G-250
- 10. Further analysis Melanie PDQuest Progenesis REDFIN • Thereare almost no possibilities to detect the appearance of a few new spots or the disappearance of single spots in large studies with several thousand spots • Evaluation of two gels by manual comparison is also impossible • It is necessary to detect differences and obtain information from gel by image collection hardware and image evaluation software • There are some 2D gel analysis software • The gels can be used for the identification and other applications by mass spectrometry
- 11. Our services At CreativeProteomics, we can provide an integrated solution for the identification of low abundance proteins in complex biological samples. Option 01 2D Electrophoresis Option 02 SDS-PAGE, IEF and native PAGE analysis Option 03 Western Blot & Electrical Transfer Option 04 2D Blue Native / SDS-PAGE for Complex Analysis
- 12.
Editor's Notes
- #2 Hello, welcome to watch Creative Proteomics’Video. Today, we are going to learn some basic knowledge about Two dimensional polyacrylamide gel electrophoresis
- #3 Two-dimensional gel electrophoresis (2-DE) is considered a powerful tool for proteomics work. It is used for separation and fractionation of complex protein mixtures from biological samples. 2-DE separates proteins depending on two differ steps: the first one is called isoelectric focusing (IEF) which separates proteins according to isoelectric points (pI); the second step is SDS-polyacrylamide gel electrophoresis (SDS-PAGE) which separates proteins based on the molecular weights(relative molecular weight, Mr). Thus, thousands of proteins can be separated, and the information about IEF and molecular weights can be obtained.
- #4 The process of 2-DE includes sample preparation, Isoelectric Focusing (IEF), SDS-PAGE, and the Visualization of results: Staining. Speaking of sample preparation, the native samples need to be converted to a physicochemical state suitable for the first dimension IEF and keep the native charge and Mr of the constituent proteins. In order to get good results, appropriate sample preparation is essential. The sample preparation are various because of the difference of the types and origins of proteins. Ideally, the process will result in the complete solubilization, disaggregation, denaturation, and reduction of the proteins in the sample.
- #5 As we mentioned above, the first dimension separate proteins depending on pI of proteins. Proteins are amphoteric molecules and the positive, negative, or zero net charge they carry depending on the pH of the surroundings. The isoelectric point (pI) is defined as the pH of a solution at which the net charge of the protein becomes zero. A protein with a positive net charge will migrate toward the cathode, becoming less positively charged until reaching its pI. While a protein with a negative net charge will migrate toward the anode, becoming less negatively charged until it also reaches its pI.
- #6 To be specific, a protein mixture is loaded at the basic end of the pH gradient gel. After applying an electric field, the proteins are separated depending on charges, focusing at positions where the pl value is equivalent to the surrounding pH. Larger proteins will move more slowly through the gel, but with sufficient time will catch up with small proteins of equal charge.
- #7 After the first dimension, the second dimension separation can be performed on flatbed or vertical systems on a slab gel. The second dimension is often performed by SDS-PAGE (SDS-polyacrylamide gel electrophoresis), which is an electrophoretic method for separating polypeptides according to their molecular weights (Mr). This method often contains four steps, including preparation of the gel, the equilibrium of the IPG strips in SDS buffer, placing the equilibrated IPG strip on the SDS gel, and finally handling the electrophoresis.
- #8 SDS can make proteins denaturing and bind to the backbone at a constant molar ratio. When applying SDS and a reducing agent (like a DTT) which can cleave disulfide bonds, proteins unfold into linear chains with negative charge proportional to the polypeptide chain length. Polyacrylamide forms a mesh-like matrix which is appropriate for separating proteins. When proteins are separated by SDS-PAGE, smaller proteins migrate faster since the less resistance.
- #9 There are various methods for visualization of proteins, but the most commonly used are Coomassie Blue staining and silver staining. Silver staining is a sensitive and non-radioactive method. The principle of silver staining is quite simple. The amino acid side chains can bind to silver ions, primary the sulfhydryl and carboxiyl groups of proteins, followed by reduction to free metallic silver. As a result, the protein bands are visualized as spots where the reduction occurs. Silver staining is suitable for low protein levels because of its sensitivity (in the very low ng range)
- #10 Coomassie Blue staining is a relatively simple method and more quantitative than silver staining. It is suitable to detect protein bands containing about 0.2 μg or more proteins. The Coomassie dye binds to proteins to form a protein-dye complex through Van der Waals attractions. There are two kinds of Coomassie dyes, R250 and G-250.
- #11 There are almost no possibilities to detect the appearance of a few new spots or the disappearance of single spots in large studies with several thousand spots. In addition, evaluation of two gels by manual comparison is also impossible. Therefore, it is necessary to detect differences and obtain information from gel by image collection hardware and image evaluation software. There are some 2D gel analysis software, such as Melanie，PDQuest，Progenesis, REDFIN etc. And the gels can be used for the identification and other applications by mass spectrometry. 2-DE is a widely used method for protein analysis with the ability to separate thousands of proteins at one time. It can provide direct visual information of changes in protein/post-translational modifications (PTMs) abundance. It can also be used to other analysis, such as whole proteome analysis, detection of biomarkers, drug discover, and so on.
- #12 At Creative Proteomics, we can provide an integrated solution for the identification of low abundance proteins in complex biological samples. We can provide two dimensional Electrophoresis, SDS-PAGE, IEF and native PAGE analysis, Western Blot and Electrical Transfer service, 2D Blue Native / SDS-PAGE for Complex Analysis
- #13 Thanks for watching our video. At creative proteomics, we provide the most reliable. If you have any questions or specific requirements. Please do not hesitate to contact us. We are very glad to cooperate with you.