Electrophoresis.pptx

Principle,instrumentation,working mechanism,advantages,limitation &application of MovingBoundary,paper,Immuno,2D,IEF, & Gel(AGE & PAGE) electrophoresis

1. Electrophoresis Dr Sumitha J,Associate Professor, JBAS COLLEGE FOR WOMEN,Chennai-18

2. Electrophoresis Electrophoresis is a technique used to separate molecules based on their charge, size, and shape. It is a powerful tool for analyzing biomolecules, such as DNA, RNA, and proteins.

3. Principle of Electrophoresis The principle of electrophoresis is based on the fact that charged particles will migrate in an electric field. The direction of migration depends on the charge of the particle: negatively charged particles will migrate towards the positive electrode (anode), while positively charged particles will migrate towards the negative electrode (cathode). The speed of migration of a particle is determined by its charge, size, and shape. Larger particles will migrate more slowly than smaller particles, and particles with a higher charge will migrate more quickly than particles with a lower charge

4. Moving boundary electrophoresis

5. MBE Moving-boundary electrophoresis is a technique used to separate charged particles based on their net charge. It is a classic method that was first developed by Arne Tiselius in the 1930s. Moving-boundary electrophoresis is based on the principle of electrophoresis, which is the movement of charged particles in an electric field.

6. Basic principles Charged particles migrate towards the oppositely charged electrode at a speed that is proportional to their net charge. The net charge of a particle is determined by its chemical composition. The isoelectric point is the pH value at which a particle has no net charge.

7. Working mechanism A sample of charged particles is placed in a solution. An electric field is applied to the solution. The charged particles migrate towards the oppositely charged electrode at a speed that is proportional to their net charge. The particles will eventually reach a point where their net charge is zero, and they will stop migrating. This point is called the isoelectric point.

8. Instrumentation A Tiselius cell A power supply A conductivity detector The Tiselius cell is a U-shaped container that is filled with a buffer solution. The buffer solution maintains a constant pH throughout the cell. The power supply provides an electric field across the cell. The conductivity detector measures the conductivity of the solution in the cell.

9. Advantages ● High resolution ● Ability to separate small molecules ● Ability to measure the isoelectric point of proteins .

10. Limitations ● Complex instrumentation ● Time-consuming ● Not suitable for large molecules

11. Applications ● Separation of proteins ● Separation of amino acids ● Determination of the isoelectric point of proteins

12. Paper electrophoresis

13. Basic principle Paper electrophoresis is a separation technique that uses an electric field to separate charged molecules. The molecules are placed on a strip of filter paper that is soaked in a buffer solution. The buffer solution helps to maintain a constant pH throughout the paper, which is important because the charge of a molecule can vary depending on the pH of the solution. When an electric field is applied, the molecules migrate towards the oppositely charged electrode. The speed at which a molecule migrates depends on its charge and size. Larger molecules migrate more slowly than smaller molecules.

14. Instrumentation The basic instrumentation for paper electrophoresis consists of a power supply a buffer tank a paper wick a electrophoresis chamber Sample applicator Detector

15. Power supply: The power supply provides the electric field that is used to separate the molecules. The voltage of the power supply is typically adjusted to be between 10 and 20 volts per centimeter. Buffer tank: The buffer tank contains the buffer solution that is used to soak the paper. The buffer solution helps to maintain a constant pH throughout the paper, which is important because the charge of a molecule can vary depending on the pH of the solution. Paper wick: The paper wick is used to transport the buffer solution from the buffer tank to the paper. The paper wick is made of a material that is able to transport the buffer solution quickly and evenly.

16. Electrophoresis chamber: The electrophoresis chamber is the container that holds the paper and the buffer solution. The electrophoresis chamber is typically a sealed container that prevents the buffer solution from evaporating. Sample applicator: The sample applicator is used to apply the sample to the paper. The sample applicator is typically a small pipette or syringe that is used to deposit a small amount of the sample onto the paper. Detector: The detector is used to detect the separated molecules. The detector can be a UV lamp or a laser scanner. The UV lamp or laser scanner is used to visualize the separated molecules on the paper.

17. Working mechanism The working mechanism of paper electrophoresis is as follows: The filter paper is soaked in the buffer solution. The sample is applied to the paper in a small spot. The power supply is turned on, which creates an electric field. The molecules in the sample migrate towards the oppositely charged electrode. The molecules are separated according to their charge and size. The migration of the molecules is stopped by turning off the power supply.

18. Advantages The advantages of paper electrophoresis include: It is a simple and inexpensive technique. It is relatively easy to perform. It can be used to separate a wide variety of molecules.

19. Limitations The limitations of paper electrophoresis include: The resolution of the separation is not as good as other methods, such as gel electrophoresis. The technique is not as sensitive as other methods. The technique is not as versatile as other methods.

20. Applications The analysis of proteins The analysis of amino acids The analysis of nucleic acids The analysis of enzymes The analysis of food colors

21. Gel electrophoresis

22. Basic principle Gel electrophoresis is a separation technique that uses an electric field to separate charged molecules. The molecules are placed in a gel that has pores of a specific size. The smaller the pores, the smaller the molecules that can pass through them. When an electric field is applied, the molecules migrate towards the oppositely charged electrode. The speed at which a molecule migrates depends on its charge and size. Larger molecules migrate more slowly than smaller molecules.

23. Instrumentation The basic instrumentation for gel electrophoresis consists of a power supply, a gel tank, a comb, and a electrophoresis chamber. The power supply provides the electric field that is used to separate the molecules. The gel tank contains the gel that is used to separate the molecules. The comb is used to create wells in the gel where the sample can be applied. The electrophoresis chamber is the container that holds the gel and the buffer solution.

24. Working mechanism The working mechanism of gel electrophoresis is as follows: The gel is prepared by mixing a polymer, such as agarose or polyacrylamide, with a buffer solution. The comb is inserted into the gel to create wells. The sample is applied to the wells. The power supply is turned on, which creates an electric field. The molecules in the sample migrate towards the oppositely charged electrode. The migration of the molecules is stopped by turning off the power supply.

25. Advantages The advantages of gel electrophoresis include: It is a very versatile technique that can be used to separate a wide variety of molecules, including DNA, RNA, and proteins. It is a very sensitive technique that can be used to detect very small amounts of molecules. It is a very reproducible technique that can be used to produce consistent results.

26. Limitations The limitations of gel electrophoresis include: It can be a time-consuming technique. It can be a destructive technique, meaning that the molecules that are being separated are destroyed in the process. It can be a difficult technique to master.

27. Applications The analysis of DNA The analysis of RNA The analysis of proteins The analysis of enzymes The analysis of food contaminants

28. Agarose Gel electrophoresis

29. AGE Agarose gel electrophoresis is a technique that uses agarose, a polysaccharide extracted from seaweed, to separate DNA fragments. The agarose gel has large pores, which allows large DNA fragments to pass through. This makes agarose gel electrophoresis a good choice for separating large DNA fragments, such as those produced by PCR.

30. PAGE Polyacrylamide gel electrophoresis is a technique that uses polyacrylamide, a synthetic polymer, to separate DNA fragments and proteins. The polyacrylamide gel has small pores, which allows only small DNA fragments and proteins to pass through. This makes polyacrylamide gel electrophoresis a good choice for separating small DNA fragments and proteins.

31. Basic principle Agarose gel electrophoresis is a technique that uses an electric field to separate DNA fragments. The DNA fragments are placed in a gel made of agarose, a polysaccharide extracted from seaweed. The agarose gel has pores of a specific size. The smaller the pores, the smaller the DNA fragments that can pass through them. When an electric field is applied, the DNA fragments migrate towards the oppositely charged electrode. The speed at which a DNA fragment migrates depends on its size and charge. Larger DNA fragments migrate more slowly than smaller DNA fragments.

32. Instrumentation The basic instrumentation for agarose gel electrophoresis consists of a power supply, a gel tank, a comb, and a electrophoresis chamber. The power supply provides the electric field that is used to separate the DNA fragments. The gel tank contains the gel that is used to separate the DNA fragments. The comb is used to create wells in the gel where the sample can be applied. The electrophoresis chamber is the container that holds the gel and the buffer solution.

33. Working mechanism The working mechanism of agarose gel electrophoresis is as follows: The gel is prepared by mixing agarose powder with a buffer solution. The comb is inserted into the gel to create wells. The sample is applied to the wells. The power supply is turned on, which creates an electric field. The DNA fragments in the sample migrate towards the oppositely charged electrode. The migration of the DNA fragments is stopped by turning off the power supply..

34. Advantages The advantages of agarose gel electrophoresis include: It is a relatively simple and easy to perform technique. It is a versatile technique that can be used to separate a wide range of DNA fragments. It is a sensitive technique that can be used to detect very small amounts of DNA. It is a reproducible technique that can be used to produce consistent results.

35. Limitations The limitations of agarose gel electrophoresis include: It can be a time-consuming technique. It can be a destructive technique, meaning that the DNA fragments that are being separated are destroyed in the process. The resolution of agarose gel electrophoresis is not as good as other methods, such as polyacrylamide gel electrophoresis.

36. Applications The analysis of DNA fragments The identification of DNA mutations The detection of DNA contamination The study of DNA structure and function

37. Polyacrylamide Gel electrophoresis

38. Basic principle Polyacrylamide gel electrophoresis (PAGE) is a technique that uses an electric field to separate proteins. The proteins are placed in a gel made of polyacrylamide, a synthetic polymer. The polyacrylamide gel has pores of a specific size. The smaller the pores, the smaller the proteins that can pass through them. When an electric field is applied, the proteins migrate towards the oppositely charged electrode. The speed at which a protein migrates depends on its size, charge, and shape. Larger proteins migrate more slowly than smaller proteins.

39. Basic principle Polyacrylamide gels are chemically cross-linked gels formed by the polymerization of acrylamide with a cross-linking agent, usually N,N’- methylenebisacrylamide . The reaction is a free radical polymerization, usually carried out with ammonium persulfate as the initiator and N,N,N’,N’- tetramethylethylendiamine (TEMED) as the catalyst.

40. Instrumentation The basic instrumentation for polyacrylamide gel electrophoresis consists of a power supply, a gel tank, a comb, and a electrophoresis chamber. The power supply provides the electric field that is used to separate the proteins. The gel tank contains the gel that is used to separate the proteins. The comb is used to create wells in the gel where the sample can be applied. The electrophoresis chamber is the container that holds the gel and the buffer solution.

41. Working mechanism The working mechanism of polyacrylamide gel electrophoresis is as follows: The gel is prepared by mixing acrylamide and bis -acrylamide monomers with a buffer solution. The comb is inserted into the gel to create wells. The sample is applied to the wells. The power supply is turned on, which creates an electric field. The proteins in the sample migrate towards the oppositely charged electrode. The migration of the proteins is stopped by turning off the power supply.

42. We use resolving and stacking gels in PAGE for two reasons: To improve resolution: The resolving gel has a smaller pore size than the stacking gel, which means that the proteins in the sample will migrate more slowly through the resolving gel. This helps to improve the resolution of the separation, meaning that the proteins can be more easily distinguished from each other. To concentrate the proteins: The stacking gel has a higher concentration of acrylamide than the resolving gel, which means that the proteins in the sample will migrate more quickly through the stacking gel. This helps to concentrate the proteins at the top of the resolving gel, which also improves the resolution of the separation.

43. Without the stacking gel, the proteins would migrate through the resolving gel at different speeds, depending on their size and charge. This would make it difficult to resolve the proteins from each other. The stacking gel helps to ensure that all of the proteins in the sample migrate through the resolving gel at the same speed, which improves the resolution of the separation.

44. Advantages The advantages of polyacrylamide gel electrophoresis include: It is a very versatile technique that can be used to separate a wide range of proteins. It is a very sensitive technique that can be used to detect very small amounts of proteins. It is a very reproducible technique that can be used to produce consistent results. The resolution of polyacrylamide gel electrophoresis is much higher than agarose gel electrophoresis.

45. Limitations The limitations of polyacrylamide gel electrophoresis include: It can be a more time-consuming and difficult technique to perform than agarose gel electrophoresis. It can be a more toxic technique than agarose gel electrophoresis. The gel is more fragile than agarose gel, so it is more difficult to handle.

46. Applications The analysis of protein fragments The identification of protein mutations The detection of protein contamination The study of protein structure and function

47. Immuno Electrophoresis

48. Basic principle Immunoelectrophoresis (IEP) is a laboratory technique that combines electrophoresis and immunodiffusion to separate and identify proteins based on their electrical charge and reactivity with antibodies. In IEP, a sample of proteins is first separated by electrophoresis in a gel. The separated proteins are then reacted with antibodies that are specific for those proteins. The antibodies and antigens will form a precipitate at the point where they meet, which can be visualized by staining the gel.

49. Basic principle Immunoelectrophoresis combines principles of electrophoresis and immunodiffusion. It involves the electrophoretic separation of proteins in a gel matrix followed by the immunodiffusion of specific antibodies against the proteins of interest. When the proteins are separated, they form distinct bands or precipitin arcs, and by adding specific antibodies, these bands can be identified and characterized .

50. Instrumentation The instrumentation required for IEP includes: An electrophoresis chamber An electric power supply A gel electrophoresis kit Antibodies specific for the proteins in the sample A staining solution

51. Working mechanism a. Electrophoresis: A mixture of proteins is loaded onto a gel and subjected to an electric field. Due to their charge-to-mass ratio, the proteins move through the gel at different rates and get separated based on their size and charge. b. Immunodiffusion: After electrophoresis, wells or troughs are cut perpendicular to the direction of protein migration in the gel. Antibodies specific to the protein of interest are added to these wells, and over time, they diffuse through the gel and form immune complexes with the corresponding proteins, creating precipitin arcs. c. Visualization: The precipitin arcs that form after immunodiffusion allow for the identification and characterization of specific proteins based on their reaction with the specific antibodies.

52. Working mechanism The working mechanism of IEP is as follows: A sample of proteins is loaded into wells in an agarose gel. The gel is placed in an electrophoresis chamber and an electric field is applied. The proteins in the gel will migrate towards the oppositely charged electrode. The proteins will be separated based on their electrical charge and size. After electrophoresis, the gel is removed from the chamber and antibodies that are specific for the proteins in the sample are added. The antibodies will diffuse through the gel and react with the antigens. The antibodies and antigens will form a precipitate at the point where they meet. The precipitate can be visualized by staining the gel.

53. Advantages The advantages of IEP include: It is a sensitive technique that can be used to detect even small amounts of proteins. It is a versatile technique that can be used to identify a wide variety of proteins. It is a relatively simple technique that can be performed in most laboratories.

54. Limitations a.Semi -quantitative: Immunoelectrophoresis is not a fully quantitative technique, and the intensity of bands may not directly correlate with protein concentrations. b. Labor -intensive: The technique can be time-consuming, particularly when analyzing multiple samples or proteins. c. Low resolution: Compared to modern techniques like Western blotting or ELISA, immunoelectrophoresis has relatively low resolution and sensitivity .

55. Applications a. Clinical diagnostics: Immunoelectrophoresis is used to detect and characterize serum proteins, including immunoglobulins, complement proteins, and acute-phase reactants, for diagnostic purposes. b. Immunology research: It is employed in various immunological studies to identify and analyze proteins involved in immune responses. c. Quality control in biopharmaceuticals: Immunoelectrophoresis is used to monitor the composition and purity of biopharmaceutical products, such as antibodies and vaccines. d. Blood banking: The technique is utilized in blood typing and antibody screening for safe blood transfusions.

56. Example An example of the use of IEP is in the diagnosis of multiple myeloma. Multiple myeloma is a cancer of plasma cells, which are a type of white blood cell that produces antibodies. In multiple myeloma, the plasma cells produce an abnormal type of antibody called a monoclonal protein. This protein can be detected by IEP.

57. IEF

58. Basic principle Isoelectric Focusing relies on the principle that charged molecules, such as proteins, will migrate in an electric field until they reach a pH at which they have no net charge, i.e., their pI . In an IEF setup, a pH gradient is created in a gel matrix, typically using immobilized pH gradients (IPG strips). Proteins are loaded onto the gel, and an electric field is applied. Proteins then move within the gel matrix towards the pH region that corresponds to their pI and become focused in sharp bands at their respective isoelectric points.

59. Instrumentation IEF can be performed using different equipment, but the most common setups include: Flatbed IEF: Where the gel is placed on a flat support and the electric field is applied using electrodes at each end of the gel. b. Capillary IEF: Where the gel is placed in a capillary tube and the electric field is applied across the length of the capillary.

60. Working mechanism a.Creating the pH gradient: This is achieved by using IPG strips, which contain a series of immobilized buffering groups with different pKa values. When an electric field is applied, these buffering groups create a stable pH gradient across the gel. b. Sample loading: The protein mixture is loaded onto the gel, usually at the acidic end of the pH gradient. c. Application of electric field: An electric field is applied across the gel, causing the proteins to migrate towards their pI . d. Isoelectric focusing: As the proteins reach their pI , they stop migrating and become focused into narrow bands.

61. Advantages a.High resolution: IEF can separate proteins with very similar charges and resolve complex mixtures with high precision. b. Sensitivity: It can detect proteins present in minute quantities. c. Sample preservation: IEF is a gentle technique that preserves protein structure and activity. d. Automation: The process can be automated, making it suitable for high-throughput analysis .

62. Limitations a.Time -consuming: IEF can be a relatively slow process compared to other techniques. b. Limited pH range: The pH range of the gel is a crucial factor, and it may not cover the pI values of all proteins of interest. c. Sample complexity: Overlapping protein bands may occur in complex mixtures, making interpretation challenging.

63. Applications a.Protein analysis: IEF is widely used for protein profiling, post-translational modification analysis, and protein purity assessment. b. Proteomics: It plays a crucial role in 2D gel electrophoresis, a common method for protein separation. c. Medical diagnostics: IEF is used in clinical laboratories to detect protein abnormalities in diseases like cancer and genetic disorders. d. Biotechnology: It is employed for protein purification in research and biopharmaceutical production..

64. 2D Electrophoresis

65. Basic principle The basic principle of 2D electrophoresis involves performing two consecutive separations on the same sample. In the first dimension, proteins are separated based on their isoelectric point using isoelectric focusing (IEF). This creates a pH gradient in a gel matrix, and proteins migrate to their respective pI positions. In the second dimension, the proteins from the first dimension gel are then subjected to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) based on their molecular weight. This results in a two-dimensional protein separation pattern, which allows for the resolution of a large number of proteins in a single gel.

66. Instrumentation The instrumentation required for 2D electrophoresis includes electrophoresis apparatus for both IEF and SDS-PAGE. Specialized equipment is needed for IEF, such as isoelectric focusing chambers and immobilized pH gradient (IPG) strips. For SDS-PAGE, standard electrophoresis apparatus and polyacrylamide gels are used.

67. Instrumentation The working mechanism of 2D electrophoresis involves the following steps: a. Isoelectric Focusing (IEF): In the first dimension, proteins are loaded onto a gel strip with a pH gradient. An electric field is applied, and proteins migrate along the pH gradient until they reach the region of the gel with a pH equal to their isoelectric point. At this point, the proteins become focused in narrow bands according to their pI . b. SDS-PAGE: The gel strip containing the focused proteins from the first dimension is then placed on top of an SDS-PAGE gel. An electric field is applied again, causing the proteins to separate based on their molecular weight. SDS denatures the proteins and imparts a negative charge, so the migration in this dimension is determined primarily by size.

68. Advantages a.High resolution: 2D electrophoresis allows for the separation of a large number of proteins, enabling the detection of minor protein variants and isoforms. b. Improved sensitivity: The two-step separation process enhances sensitivity, especially for low-abundance proteins. c. Identification and characterization: The separated proteins can be further analyzed using various staining methods or transferred to a membrane for Western blotting or mass spectrometry-based identification.

69. Limitations a.Labor -intensive: 2D electrophoresis can be time-consuming and requires careful handling of gels to prevent artifacts. b. Limited dynamic range: The detection of very high-abundance and very low-abundance proteins in the same gel may be challenging due to the limited dynamic range of the technique. c. Protein modifications: Post-translational modifications, such as glycosylation, may affect protein migration and complicate the interpretation of results.

70. Applications a.Proteomics : 2D electrophoresis is a key tool in proteomics research for protein profiling and identifying changes in protein expression under different conditions. b. Disease biomarker discovery: It is used to identify potential biomarkers associated with diseases like cancer and neurological disorders. c. Comparative analysis: 2D electrophoresis is employed to compare protein profiles between different samples or experimental groups to study disease mechanisms or drug effects.