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Page electrophoresis 08_aug12
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Page electrophoresis 08_aug12

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  • 1. Quality Control of Product Polyacrylamide Gel Electrophoresis
  • 2. Analysis of Product • Quality Control involves the entire process of obtaining a product that meets defined specifications expressing both its purity and potency • Testing methods include cell biology, virology, chemistry, analytical chemistry, molecular biology & the potency of the product • Different methods have different levels of detection ie, values can go from grams to nanograms
  • 3. Electrophoresis and Movement of Molecules • Molecules can have distinct charges – Positive or Negative – Net charge will cause different movement through gel • Molecules can have different shapes – Linear – globular – Alpha helix +
  • 4. Macromolecular charge • Macromolecules have a variable net charge that depends on pH • pH at which net charge is zero = pI • Electrical shielding of charge occurs when counterions are solvated V= V =
  • 5. Electrophoresis • Horizontal Agarose Gels • Agarose forms a gel or molecular sieve that supports the movement of small materials in solution used for DNA • Vertical Polyacrylamide Gels • Made of Polyacrylamide • Used for Protein molecular size, shape, charge • IEF electrophoresis • Western Blot technique
  • 6. Horizontal Gels • Gel Box set up frequently used in DNA analysis
  • 7. Agarose gels • Usually used in DNA analysis • Made up of linear polysaccharide mol wt of 12,000 • Basic repeating unit is agarobiose • Gels are prepared at 1% to 3% providing tunnels for molecules to move through • DNA can be much larger then most proteins
  • 8. Agarose Gel with DNA Bands • DNA is negatively charged • Smaller sized DNA moves faster than Larger DNA • Markers are used to determine relative sizes of DNA pieces markers
  • 9. PAGE • Native : Protein is prepared with little disturbance to the cellular material – Proteins are associated – Movement of samples through the gel can be inconsistent • SDS : Sodium Dodecyl Sulfate Is a detergent – Protein coated with a negative charge in proportion to its molecular weight – Denatures and unfolds protein – Reducing agents (DTT)break amino acid cross-links
  • 10. P Polyacrylamide Gel Creates tunnels in gel for molecules to move through
  • 11. Uses for PAGE • Separates proteins from each other – Proteins separated by size – Isoelectric point • Determines – Molecular size of protein – Quantifies the amount present – Displays Impurities – Used in western blot assays by antigen interactions
  • 12. Determine Molecular Weight 1. Run standard molecular weight markers on gel 2. Run unknown protein on the same gel 3. Create a graph of the mol wt versus distance molecule has moved 4. Using the distance the unknown has moved determine the molecular weight from graph
  • 13. Molecular Weight Markers Migration of molecular weight of standards are compared to unknown samplewt std vs unknown
  • 14. Molecular Weight vs Distance
  • 15. Western Blot Analysis • Identifies protein through antibody interaction • Run proteins on denatured gel (SDS-PAGE) • Transfer (blot) proteins onto membrane • Probe the membrane with primary antibody • Add secondary antibody (this antibody is linked to an enzyme) • Substrate is added and color appears
  • 16. SDS Polyacrylamide Electrophoresis
  • 17. SDS Effect on Protein Movement • Sodium Dodecyl Sulfate denatures protein and covers it with negative charges : moves to + end • Vertical gels are designed so the top of the gel box is attached to the negative power outlet • The bottom of the gel box is attached to the positive power outlet • Movement through the PAGE gel is proportional to mass not to charge
  • 18. Movement of Proteins on an SDS Gel Stacking of proteins at top of gel at start Low weight molecular dye - + Distribution of proteins in a charged field Protein Migration Highest Molecular Wt. protein
  • 19. % Polyacrylamide in Gel • Gels can be made at different concentrations of polyacrylamide • Example: gels made at 3%,6%,9% and 12% will produce different openings through which the molecule will migrate • The larger the opening allows large molecules to move through the gel
  • 20. Vertical Polyacrylamide Gel Electrophoresis
  • 21. Equipment for Electrophoresis
  • 22. Gel Electrophoresis Equipment Mini-PROTEAN Tetra Cell
  • 23. Closed Mini Gel holder
  • 24. Open Gel Holder: Allows New Gel to be Inserted
  • 25. Gel Holders Placed in Mini-Protean Tetra Cell
  • 26. Procedure in Short LoadGe Place Buffer Equip
  • 27. Electrophoresis of Samples Setting Up and Running Mini-PROTEAN TGX Precast Gels – Youhttp://www.youtube.co m/watch?v=XnEdmk1SqvgT ube Samples: boiled 3’ with loading dye (2x Laemmli buffer + running dye) Mini-PROTEAN tetra cell: Set up according to SOP given in workbook Power settings: 75 volts for 45 – 60 minutes Running dye should not run off the bottom of gel

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