Cell Bio 1

  1. 4 pillars of biology
    biochem, microscopy, genetics, and cell biology
  2. Key features of the cell
    everything is dynamic, diffusion, spatial organization
  3. when did cellular life come to exist
    3.5 billion yrs ago
  4. when did eukaryotes come to exist
    endosymbiotic event occurred 1-2 billion yrs ago
  5. central dogma
    DNA transcription RNA translation protein
  6. about how many genes are in the human genome
    20,000 genes
  7. about how many proteins are in the cell
  8. size of prokaryotes
    1-2 microns
  9. size of eukayotes
    1-100 microns
  10. function of the smooth ER
    produce lipids
  11. purpose of the rough ER
    common in cells that export proteins
  12. structure of the cell membrane
    50% phospholipids and 50% proteins
  13. purpose of membranes
    it exists for the purpose of organization
  14. purpose of an enzyme
    makes reactions more kinetically favorable and provides specificity
  15. size of protein
  16. large dissociation constant
    weak interaction between A and B
  17. low dissociation constant
    strong interaction between A and B
  18. interaction between two actin monomers
    relatively low interaction
  19. describe protein-protein interaction
    • in cells they are relatively weak individually, but collectively strong
    •  ex: actin monomers
  20. what can make reactions more favorable
    higher concentration of substrates and enzymes
  21. difference between the dynamics of small and large molecular structures
    • large scale things move very slowly throughout the cell and small scale things move quickly (chaotic movements)
    • really large scale things (i.e. mitochondria and ribosome can not move unless an outside force acts on it)
  22. how do enzymes work
    they stabilize the intermediate of the reaction to lower the activation energy
  23. old processes (RNA world) vs "new" processes
    ribosomes (RNA catalysis), everything started with RNA

    everything is pretty much ran by proteins
  24. Details of DNA
    • chemically relatively inert
    • structurally relatively boring
    • great information storage
  25. Details of proteins
    • relatively labile
    • don't last long
    • highly structured (cool structures)
    • not good at information storage
    • catalytic
  26. Details of RNA
    • decent information storage
    • so-so catalytic 
    • chemically labile
    • short lived
  27. protein secondary structures and bonds
    • hydrogen bonds
    • alpha helix and beta sheets
  28. how many alpha AA per alpha helix turn
  29. average number of an amino acid in a protein
  30. size of an amino acid
    110 Da per AA
  31. tertiary structure of proteins
    • folded and connected alpha and beta sheets and linkers.
    • folding can be lead by hydrogen bonding and hydrophobic affects
  32. differential centrifugation
    separate something based on solubility
  33. SDS-Page Gel
    Sodium dodecyl sulfate polyacrylamide gel electrophoresis
  34. function of electrophoresis
    • separates Proteins based on size
    • protein is negative and the gel runs from positive to negative
  35. how to denature a complex protein
    • 1st step- increase temp, change pH, or change [urea]
    • 2nd step- add SDS to coat the protein string
  36. steps for separating protein in SDS-Page
    • protein added to gel and electrical charge runs from top to bottom
    • stain gell with coomasie blue 
    • destain to see bands
  37. type of column chromatography
    gel filtration, ion exchange, and affinity matrix
  38. how does gel filtration work
    • separates protein based on size
    • beads are chemically inert
    • beads have holes in them 
    • smaller proteins go through the holes 
    • big proteins pass through
  39. how is the size of  protein determined
    by its stokes radius
  40. what is stokes radius
    biophysical radius of a molecule in solution
  41. Ion exchange Column
    • separates based on charge
    • add low salt solution and then increase salt concentration
    • proteins are extracted from positive to negative
  42. affinity chromatography
    • separates protein based on biochemical affinity
    • beads contain antibodies that bond to specific proteins
    • change ph to unfavorable conditions to elude protein
    • last fraction should contain pure protein
  43. steps for Western Blot
    • 1-block non-specific binding with milk and or BSA
    • 2- add primary antibody to bind to desired protein
    • 3- add secondary antibody that may contain an enzyme, fluorophore
  44. how do fluorophores work
    • fluorophores absorb a photon and later emit photon of a different color
    • photon emitted has a lower energy than the photon absorbed
    • dark to light
  45. 4 main parts of microscopy
    • resolution, contrast, magnification, sample preparation
    • there's usually a tradeoff
  46. resolution
    differentiation between two different objects
  47. contrast
    difference between different colors
  48. Bright field microscope
    • some light passes through the sample, while light that hits an object doesn't pass through
    • not the best contrast and its cheap
  49. Phase contrast
    • dark and light
    • molecules in the cell have different refractive indexes
    • specific light paths are delayed and causes destructive interference (dark spots)
    • useful to look at large cells
  50. epifluorescence microscopy
    primary antibodies attacked to secondary antibodies containing a fluorophore
  51. know how the epi microscope works
    • filter on white light only send blue light through microscope
    • blue light hits fluorophore and emits green light
    • microscope filters out all the blue light
  52. limitations of EPI
    have to kill cells to get antibody in the cell
  53. GFP
    • green fluorescent protein
    • can be modified to express different colors
    • is place in a sequence near the sequence of the protein
    • placed in epifluorescence microscope
  54. superresolution microscopy
    • can alternate the fluorescence emission of a fluorophore really close to each other to increase resolution
    • 10-fold resolution increase
  55. drawbacks of superressolution microscopy
    • really hard and expensive to do
    • cells die when you shine a bright light on them for long periods of time
  56. Electron microscope
    • shoots electrons at the samples
    • two types: SEM and TEM
    • resolution is limited by the sample not the microscope
  57. SEM
    • scanning electron microscopy
    • shows outside surface of things
    • sample is covered in thin metal stain
    • e- can bounce off the surface
    • sample prep is toxic, kills the cells
    • microscope operates of under a vacuum
  58. TEM
    • transmission electron microscopy
    • see the insides (x-ray)
    • electrons are shot and a picture is made based on the electrons that shoot through and are deflected
    • high resolution
  59. drawbacks of TEM
    sample prep is harsh
  60. improvements for TEM and drawbacks of improvement
    • add antibodies with gold  (immunogold) that bind to specific proteins
    • microscope can identify gold piece
    • sample prep is harsh and could change the orientations of organelles and proteins in the cell
  61. light sheet microscopy
    • variation of epifluorescence
    • quickly shine light through a small sliver of the sample and take a picture from above 
    • stack layers to create a 3D image
Card Set
Cell Bio 1
Exam 1