2D Gel Electrophoresis

2D gel electrophoresis is a widely used technique in molecular biology and biochemistry to separate and analyze complex mixtures of proteins. It combines two dimensions of separation, isoelectric focusing (IEF), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), to achieve high-resolution separation of proteins based on their isoelectric point (pI) and molecular weight.

Here is a step-by-step overview of the 2D gel electrophoresis process:

1. Sample Preparation: The first step involves extracting proteins from the biological sample of interest. The sample can be a cell lysate, tissue extract, or any other protein-containing mixture. The proteins are typically solubilized and denatured using a lysis buffer containing detergents and denaturing agents.

2. Isoelectric Focusing (IEF): The next step is to perform the first dimension separation, which separates proteins based on their pI. In IEF, proteins are loaded onto an immobilized pH gradient (IPG) gel strip or a strip of carrier ampholytes with a pH gradient. An electric field is applied across the strip, causing the proteins to migrate toward their respective pI, where they become electrically neutral and stop moving. The separation occurs in a tube gel or a flat gel format.

3. Equilibration: After the completion of IEF, the IPG strip is equilibrated to prepare it for the second dimension separation. This involves treating the strip with reducing and alkylating agents to ensure proper SDS-PAGE separation and to prevent protein aggregation.

4. SDS-PAGE: In the second dimension, the equilibrated IPG strip is placed on top of an SDS-PAGE gel, which is typically a polyacrylamide gel with a concentration gradient. The proteins are separated based on their molecular weight as an electric field is applied across the gel. SDS, a detergent, denatures the proteins and imparts a negative charge to them, allowing for separation based on size. The proteins migrate through the gel, with smaller proteins moving faster and larger proteins moving more slowly.

5. Visualization and Analysis: After the electrophoresis run, the proteins are typically stained using specific dyes, such as Coomassie Brilliant Blue or silver stain, to visualize the protein bands. The gel can be scanned or photographed for documentation and further analysis. Advanced techniques like mass spectrometry can be used to identify individual proteins within the gel spots/bands.

Overall, 2D gel electrophoresis allows researchers to obtain a two-dimensional map of the protein composition within a sample, facilitating the detection of differences in protein expression, post-translational modifications, and protein-protein interactions. It has been a valuable tool in various fields, including proteomics, biomedical research, and biomarker discovery.

1. 2 D GEL ELECTROPHORESIS Presented by: ISHWAR PATIL REGULATORY TOXICOLOGY M.S.PHARM/2022-24/RT/10

2. LEARNING OBJECTIVES Understanding of gel electrophoresis and its uses. Types of gel electrophoresis Limitations of gel electrophoresis. Understanding of 2D gel electrophoresis Steps involved in 2D gel electrophoresis Its applications.

3. 2D GEL ELECTROPHORESIS

4. PRINCIPLE Electrophoretic separation is based on the migration of unbalanced charged molecules in an electric field and is the most frequently used dispensation method in the study of proteins and nucleic acids . The main premise of electrophoretic separation is application of an electric field that forces molecules to move through gel pores, separating them based on their MW and total particle charge. Large-molecular weight molecules are slowed down on the basis of gel pore size, more specifically, larger-molecular-weight molecules are “ trapped ” in regions of the gel with a higher percent concentration .

5. Housing and feeding conditions Gel electrophoresis -Horizontal or vertical orientation   H orizontal gels - agarose matrix . V ertical gels - acrylamide matrix . Pore sizes of these gels depend on the concentration of chemical components. A garose gel pores (100 to 500 nm diameter) are larger and less uniform compared to that of acrylamide gel pores (10 to 200 nm in diameter). Comparatively, DNA and RNA molecules are larger than a linear strand of protein , which are often denatured prior to, or during this process, making them easier to analyze. Thus, DNA and RNA molecules are more often run on agarose gels ( horizontally ), while proteins are run on acrylamide gels ( vertically ).

6. Uses Of Gel Electrophoresis It is a technique used for the separation of Deoxyribonucleic acid, Ribonucleic acid or protein molecules according to their size and electrical charge using an electric current applied to a gel matrix.     Gel is a cross linked polymer whose composition and porosity is chosen based on the specific weight and porosity of the target molecules.     Types of Gel: ▪ Agarose gel. ▪ Polyacrylamide gel.  

7. Electrophoresis in a single dimension is useful for separation of few proteins simultaneously but large number of proteins can not be separated with good resolution. Complex mixtures e.g. serum, cell lysate can’t be separated. Need technique to provide better resolution at proteome level. LIMITATIONS OF 1D ELECTROPHORESIS

8. Separation and identification of proteins in a sample by displacement in 2 dimensions oriented at right-angle to one another.   First dimension : Separates proteins pH on gradient based on isoelectric point ( pI ) using isoelectric focusing   Second dimension : Following IEF, proteins are resolved according to their molecular weight using SDS-PAGE   2D Gel Electrophoresis

9. WORK FLOW OF 2D ELECTROPHORESIS Isoelectric focusing (first dimension) Equilibration of IPG strips SDS-PAGE (second dimension) Staining – gel visualization Image analysis Spot picking Enzymatic digestion MS analysis

10. Protein separation according to isoelectric point P roteins introduced into immobilized pH gradient Electric field is applied in which protein migrates according to its charge Protein reaches Isoelectric point (pI) pH = pI protein does not move in electric field owing to the lack of charge ISOELECTRIC FOCUSING

11. Rehydration Rehydrate IPG strips overnight in a re swelling tray at RT using solution containing the extracted protein in buffer (rehydration/IPG buffer) Passive rehydration – no voltage applied Active rehydration – apply low voltage Overlay mineral oil on rehydrated strips IPG plates used for IEF IPG strips different pH ranges (e.g. pH4-7, 3-10 etc) IPG strips length are between 7-24 cm. IEF units are capable of accommodating IPG strips of different length (7-24 cm). Large gels are recommended to resolve spots better. However, handling large gels is tedious 2. EQUILIBRATION OF IPG STRIPS

12. SDS PAGE Equilibrating IPG strips after IEF . Applying IPG strips to the 2D SDS PAGE. Performing SDS PAGE. REMOVE THE IPG STRIPS FROM THE TRAY SDS PAGE : Sodium dodecyl sulfate poly acrylamide gel electrophoresis. SDS is a detergent and used to give a negative charge to denatured protein. One molecule of SDS binds every 2 amino acids of protein. PLACE THE IPG STRIP FACING UP IN THE EQUILIBRATION BUFFER

13. IPG strip is placed on top of the pre-cast SDS-PAGE gel and electric current apply

14. Staining And Gel Visualization

15. Image Analysis Separation on basis of molecular weight not isoelectric point. Requires modest voltage. Requires a shorter period of time. Presence of SDS is critical to disrupting structure and making mobility. Degree of resolution determined by percentage of acrylamide and electric field strength.

16. Analysis Identification of eluted proteins spot MALDI - TOF (Matrix Assisted Laser Desorption/Ionization -Time of flight) MS (Mass Spectroscopy) Peptide Mass Fingerprint

17. Applications Analyzing proteome profiles. Detecting post- or co-translational modifications. Discovering new drug targets. Studying protein expression in normal, disease, or developmental states. Identifying novel proteins.

18. A. Drabik and A. Bodzon-Kułakowska ; J. Silberring ; GEL ELECTROPHORESIS; Proteomic Profiling and Analytical Chemistry; Proteomic Profiling and Analytical Chemistry. http://dx.doi.org/10.1016/B978-0-444-63688-1.00007-0 2016 Elsevier Sameh Magdeldin , Shymaa Enany , Yutaka Yoshida , Bo Xu , Ying Zhang , Zam Zureena , Ilambarthi Lokamani , Eishin Yaoita and Tadashi Yamamoto; Basics and recent advances of two dimensional- polyacrylamide gel electrophoresis; Magdeldinetal. Clinical Proteomics 2014 References

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