Electrophoresis...

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Electrophoresis...

  1. 1. Electrophoresis Aneeqa Naseer 2011-ag-3886 Ayesha Javed 2011-ag-3889 Sadia Manzoor 2011-ag-3944
  2. 2. Contents: 1) Why electrophoresis?? 2) What is electrophoresis?? 3) Principle 4) Agarose gel electrophoresis 5) Sds-PAGE (Polyacrylamide gel electrophoresis) 6) Factors effecting electrophoresis 7) applications
  3. 3. Why electrophoresis?  To separate DNA fragments from each other  To determine the sizes of DNA fragments  To determine the presence or amount of DNA  To analyze restriction digestion products.  Determination of molecular weight of proteins.
  4. 4. What is electrophoresis?  It describes migration of charged particles or molecules under the influence of electric field.  It is standard method for separation, identification, analysis and purification of: –DNA molecules –protein molecules
  5. 5. PRINCIPLE  separates molecules from each other on the basis of –size –charge –shape  basis of separation depends on how the sample and gel are prepared?
  6. 6. Gel electrophoresis  What is a gel? 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. 7. Agarose gel electrophoresis Agarose gel:  Agarose is extracted in the form of agar from several species of red marine algae, or seaweed, found in California and eastern Asia, dissolves in near-boiling water, and forms a gel when cools.  Has gelling temperature 35-38˚C and melts at 90-95 ˚C.  Used to separate macromolecules such as nucleic acids, large proteins and protein complexes. Composition:  It is prepared by dissolving 0.5% agarose in boiling water and allowing it to cool to 40°C.  It is fragile because of the formation of weak hydrogen bonds and hydrophobic bonds.
  8. 8. Material required for gel electrophoresis  Electrophoresis chamber  Agarose gel  Gel casting tray  Buffer  Staining agent (dye)  A comb  DNA ladder  Sample to be separate
  9. 9. Electrophoresis Equipment
  10. 10. Gel Casting Trays  available in a variety of sizes and composed of UV-transparent plastic.  The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis.
  11. 11. Applied voltage •  voltage,  rate of migration • The higher the voltage, the more quickly the gel runs • But if voltage is too high, gel melts • The best separation will apply voltage at no more than 5V/cm of gel length.
  12. 12. Buffers  During electrophoresis water undergoes hydrolysis : H2O  H + OH-  Buffers prevent the pH from changing by reacting with the H+ or OH- products  Most common buffer used is called TRIS – [tris (hydroxymethyl) aminomethane]
  13. 13. Buffers (cont.)  Another compound is added to make Tris an effective buffer — either boric or acetic acid  Another compound is added to bind metals EDTA  The buffer is either TBE or TAE  TBE is made with Tris/Boric Acid/EDTA  TAE is made with Tris/Acetic Acid/ EDTA
  14. 14. Staining of DNA  To make DNA fragments visible after electrophoresis, the DNA must be stained  The favorite—ethidium bromide  When bound to DNA it fluoresces under ultraviolet light (reddish –orange color)  Convenient because it can be added directly to the gel  Sensitive—detects 0.1ug of DNA.  ethidium bromide is mutagenic so care must be taken while handling the dye.  Other alternatives for ethidium bromide :  Methylene blue  Syber safe  xylene cyanol  bromphenol blue
  15. 15. A Comb  A comb is placed in the liquid gel after it has been poured  Removing the comb from the hardened gel produces a series of wells used to load the DNA
  16. 16. DNA ladder  It is a solution of DNA molecules of different length  DNA Ladder consists of known DNA sizes used to determine the size of an unknown DNA sample.  The DNA ladder usually contains regularly spaced sized samples which when run on an agarose gel looks like a "ladder".
  17. 17. Method For Electrophoresis Add running buffer, load samples and marker Run gel at constant voltage until band separation occurs Pour into casting tray with comb and allow to solidify View DNA on UV light box and show results Prepare agarose gel Melt, cool and add Ethidium Bromide. Mix thoroughly.
  18. 18. Get your sample obtained from previous purifying technique (i.e. PCR) Set up gel, remove comb Load Buffer Load Sample Run Gel Stain and look at with UV light
  19. 19.  DNA is negatively charged. +- Power DNA  When placed in an electrical field, DNA will migrate toward the positive pole (anode). H  O2   An agarose gel is used to slow the movement of DNA and separate by size.
  20. 20. SDS  Since we are trying to separate many different protein molecules of a variety of shapes and sizes, we first want to get them to be linear so that the proteins no longer have any secondary, tertiary or quaternary structure.
  21. 21. SDS (cont.)  To have proteins with linear structures we use sodium dodecyl sulfate (SDS).  SDS is a detergent that can dissolve hydrophobic molecules, also have negative charge (sulphate) attached to it allowing it to run properly through the gel (from negative to positive).  If cell is incubated with it o membranes will be dissolved o Proteins will be solubilized by it o All proteins covered with negative charges
  22. 22.  End result has two important features: 1. Proteins contain only primary structures. 2. In electric field migrate towards positive pole.
  23. 23. Why PAGE?  Proteins separate only on the basis of charges in electric field.  We also need to separate it on basis of size.  To separate it on basis of size we use PAGE.
  24. 24. It is polymer of acrylamide monomers when its polymer is formed it turns into gel and we use electricity to pull proteins through the gel, process is called polyacrylamide gel electrophoresis. Polyacrylamide gel Tunnels of different diameters What is PAGE?
  25. 25. Polyacrylamide gel  Polyacrylamide is a polymer of acrylamide monomers.  Like Agarose Gels, Polyacrylamide gels are used to separate protein molecules by shape, size and charge.  Polyacrylamide is specifically used for proteins because it provides the protein with an environment where it will not become denatured.  Allowing different sized proteins to move at different rates.
  26. 26. Polyacrylamide gel  When electricity passed proteins tend to move through gel in bunches, or bands, since there are so many copies of each protein and they are all same size.  Controlled time is given for run so that proteins not reached to other side of the gel.  After it stain proteins.  Bands
  27. 27. SDS-PAGE
  28. 28. Gel has 5 number lanes where 5 different samples of proteins are applies to gel. Lane 1: molecular weight standards of known sizes. Lane 2: mixture of 3 proteins of different sizes with a being biggest and c being smallest. Lane 3: protein a by itself Lane 4: protein b by itself Lane 5: protein c by itself
  29. 29. Factors affecting electrophoresis • 1.The sample • 2. DNA or RNA Molecular Weight • 3. Voltage • 4. Agarose • 5. Buffer • 6. Visualisation
  30. 30. The Sample: Charge/mass ratio of the sample dictates its electrophoretic mobili ty. The mass consists of not only the size (molecular weight) but also the shape of the molecule. • Charge: The higher the charge, greater is the • electrophoretic mobility. The charge is dependent on pH of the medium. b) Size: The bigger molecules have a small electrophoretic mobility compared to the smaller particles. c) Shape: The globular protein will migrate faster than the fibrous protein
  31. 31.  DNA or RNA Molecular Weight The length of the DNA molecule is the most important factor, smaller molecules travel farther.  voltage The higher the voltage, the faster the DNA moves. But voltage islimited by the fact that it heats and ultimately causes the gel to melt. High voltages also decrease the resolution (above about 5 to 8 V/cm)
  32. 32. Agarose • Agarose gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pair to several megabases (millions of bases) using specialized apparatus. Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. • The distance between DNA bands of a given length is determined by the percent agarose in the gel. In general lower concentrations of agarose are better for larger molecules because the result in greater separation between bands that are close in size. The disadvantage of higher concentrations is the long run times (sometimes days). Instead high percentage agarose gels should be run with a pulsed field electrophoresis (PFE), or field inversion electrophoresis.
  33. 33. Buffer • The most common buffers for agarose gel: TAE: Tris acetate EDTA TBE: Tris/Borate/EDTA SB: Sodium borate. • TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product. visualization Ethidium bromide dye which is used as staining dye, in staining is carcinogen.
  34. 34. applications • Vast applications include i. vaccine analysis ii. DNA analysis iii. Protein analysis iv. Antibiotic analysis
  35. 35. Vaccine analysis • Electrophoresis is widely used in vaccine analysis method. • Several vaccines have been purified, processed and analysed. e.g. influenza vaccine, polio vaccine, hepatitis vaccine.
  36. 36. DNA analysis • Through electrophoresis specific DNA sequences can be analysed, isolated and cloned. • Analysed DNA may be used in forensic investigations and paternity tests.
  37. 37. Protein analysis • Electrophoresis has advanced our understanding on the structure and function of protein. • Amount of protein in your urine and blood is measured and compared to established normal values- lower or higher than the normal levels usually indicates a disease.
  38. 38. Antibiotic analysis • . With electrophoresis, experts are not only able to synthesize new antibiotics but are also able to analyze which types of bacteria are antibiotic-resistant. • These drugs, such as penicillin, are among the widely prescribed drugs against bacterial infections

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