Electrophoresis..pptx

Basic Principle, working mechanism, instrumentation,advantages,limitations and applications of Electrophoresis
Moving boundary
Paper
AGE & PAGE

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 D etector

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. 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.

36. 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.

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

38. Polyacrylamide Gel electrophoresis

39. 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 .

40. 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.

41. 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.

42. 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.

43. 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 .

44. 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.

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