1.25.2010 lecture


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1.25.2010 lecture

  1. 1. 1.25.2010 lecture<br /><ul><li>Review of previous lecture:
  2. 2. Graph between human cells and mouse cells in cultures.
  3. 3. Confluency is when cells have divided where they are now all in contact with another. The cells enter senescence.
  4. 4. Signals can shut down the division of cells.
  5. 5. Senescence/aging – phase two, a stage of life for the cell where mitosis ceases and the cell is at rest, or in a period of aging
  6. 6. What causes this stage? Telomere shortening at each round of cell division.
  7. 7. Cells divide from 50-100 times.
  8. 8. Hafflick limit – # of times a cell divides and then cell senescence starts
  9. 9. Some cells in the mouse crisis phase avoid cell death, continue to divide in the culture. Transformed cells that divide in an unregulated way. They become cell lines.
  10. 10. Apoptosis – after senescence, cells die, or programmed cell death.
  11. 11. Microscopy Sections Review
  12. 12. Light microscopy
  13. 13. Formula for resolution
  14. 14. Brightfield
  15. 15. DIC Imaging
  16. 16. Phase Contrast Imaging
  17. 17. When white light is passed through cell, it’s hard to see much because of transparency, so you can use DIC or phase contrast.
  18. 18. Phase Contrast - Two waves of light in phase increases intensity of the light and you are able to see better if disrupted, that creates contrast to be visualized by eye.
  19. 19. DIC – disrupts light but put a prism in microscope that bends light to create a bright halo around organelles and vesicles and plasma membrane. Usually used to visualize vesicle transport. Allows you to image live cells.
  20. 20. Antibodies Used in Culture
  21. 21. Immunoglobulin proteins
  22. 22. Contain immunoglobulin regions – allow antibodies to interact with pathogens or proteins. Antibodies have sites for lipids too etc.
  23. 23. Heavy chains linked by disulfide bonds (cysteine)
  24. 24. Variable region (near amino terminus)
  25. 25. Antigen binding site is the region of the antibody that will recognize the protein of interest trying to visualize.
  26. 26. Normally, it’s targeted for a particular component of a pathenogenic organism or cell-cell.
  27. 27. Constant region (whole tale of the antibody)
  28. 28. Epitopes – epitope binding sites. A single antibody recognizes a single epitope on a protein or bacteria. Some epitopes induce an immune response if the region on the protein/bacteria is immunogenic, others will not and those are called antigenic.
  29. 29. A single b cell has a single antibody as well as a single epitope specificity. They are expressed in the membrane.
  30. 30. Polyclonal antibodies
  31. 31. Generated in rabbit (usually). Subcutaneous injections, blood collected, centrifuged, separated serum containing antibodies.
  32. 32. Have every possible antibody that can bind to multiple epitopes on that protein of interest.
  33. 33. Less specific (cross between other eipitopes)
  34. 34. Cheaper
  35. 35. Quicker to generate
  36. 36. Monoclonal antibodies
  37. 37. Antibodies that have a single specificity for one epitope on that protein.
  38. 38. Mouse
  39. 39. Subcutaneous injections, take b cells from spleen that have been making antibodies and fuse with cells that grow well in culture (an onogenic cell line)
  40. 40. You result in a heterokaryon, or a hybridoma cells
  41. 41. Can make antibodies for one epitope
  42. 42. Can continually divide
  43. 43. More specific
  44. 44. Constant source of antibody, so cheaper in that way
  45. 45. Fluorescently tagged antibodies (usally gold)
  46. 46. E- microscope allows you to look at gold plarticles within cell walls and see the pectin molecules due to the gold attached to the pectin antibody.
  47. 47. Look at a protein that is expressed
  48. 48. Western blotting
  49. 49. Taking all proteins in cell, removing them from cell by collecting them all, and running on a polyacrylimide gel, separating by size.
  50. 50. Coomassie stained gels – directly staining the gel to observe separated proteins.
  51. 51. Take a filter to probe with antibody of interest and use to detect whether or not the protein it recognizes is there.
  52. 52. Can use them to bind proteins to see what happens if protein doesn’t function.</li></ul>A secondary antibody is an antibody that binds to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection, purification or cell sorting applications.<br />Secondary antibodies may be polyclonal or monoclonal, and are available with specificity for whole Ig molecules or antibody fragments such as the Fc or Fab regions.<br />Specific secondary antibodies are usually chosen to work in specific laboratory applications. Frequently, any one of several secondary antibodies perform adequately in a particular application. They are selected according to the source of the primary antibody, the class of the primary antibody (e.g., IgG or IgM), and the kind of label which is preferred. Identifying the optimal secondary antibody is normally done through trial and error.<br />