Molecular Biology 5 - Separating Molecules

MolBio 5 pt 1

MolBio 5 pt 2

Learning Outcomes Pt 1

• Know the different Gel compositions
  • Agarose - polysaccharide mesh - lower resolution
  • Polyacrylamide - crosslinked - more uniform, higher resolution. Needed when separating many (hundreds) of molecules.
• Know the basis of separation in Gel Electrophoresis is by:
  • Charge - determines direction: Negative Molecules move down to positive electrode
  • Size - determine distance: smaller molecules move faster / lower
  • shape - supercoiled DNA moves faster / lower than expected
• Know typically gel percentages
  • 1% standard
  • higher for smaller molecules
  • lower for bigger molecules
• Know that running uncut is useful for comparison / to know where supercoiled DNA runs
• Know that ladders / MW standards are to gauge size
  • be able to estimate concentration given ladder band concentrations
• Know how to visualize DNA
  • Ethidium Bromide (EtBr) + UV
  • know that Ethidium is positive and will migrate up
  • know the hazards of EtBr
• Know that gel loading dye helps to load
  • glycerol (not in videos) to weight sample down
  • any dye to see gel being loaded
  • usually negative dye to help see migration front

Terms:

• comb
• well
• lane
• band(s)
• coiling / supercoiling
• anode = negative electrode
• cathode = positive electrode

Learning Outcomes Pt 2

• Know that centrifugation / spinning separates by density
• Know that centrifuges MUST be balanced
  • given a diagram, be able to balance the centrifuge
  • know that unbalanced centrifuges make noise
  • know that you shouldn't walk away from a centrifuge until it has reached speed
• Know that special tubes may be required
• Know that chromatography separated based on affinity / interactions
• Know the 2 phases
  • mobile phase containing mixture that is to be separated
  • stationary phase - contains some element / material that retains parts of the mobile phase mixture
• Be able to give a few examples of chromatography and the basis of separation.
  • paper: by solubility
  • 2D paper: by solubility
  • Thin-Layer (TLC): by solubility & interaction with plate material
• Understand that in liquid chromatography a liquid mixture is passed through a column of fixed material (that will bind the desired component)
• Be able to give example of how the liquid is passed through
  • gravity
  • centrifugation
  • pumps / vacuum (ex: syringe)
    • HPLC = Higher Performance Liquid Chromatography using higher pressure and small sample size
• Know that ion exchange column works by binding a molecule of opposite charge, then disrupting that charge (eluting)
• Be able to give example of other types of liquid chromatography columns and what they might bind
  • substrate: to bind enzyme
  • ligand: to bind receptors
  • antibody: to bind antibody target
• Know that a "Tag" is protein engineered to be "fused" to a target protein. A recombinant protein. 
• Know that Tag proteins are useful in fractionations because a common column can be used for many different targets.
• Give examples of tags and how they are targets
  • Column with Zn2+ (or other metal ion) to bind Poly-His tag
  • Glutathione column - bind GST tage
  • anti-Flag / anti-Myc / anti-HA columns will bind Flag, Myc, or HA tags
• Know that Gas chromatography, Mass Spec and HPLC are use more for identification of components that for purification
• Know that flow cytometry uses antibodies with a fluorescent dye is used to mark cells, which can then be counted
• Know that FACS (fluorescence activated cell sorting) can then be used to sort cells that have been fluorescently marked
• Know that filtration (and dialysis) is by size
• know that filtration is a common technique for sterilization

Terms:

• supernatant
• pellet
• fixed-angle vs swinging / bucket
• Elution
• Flow-through / wash

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The Gel extraction / liquid chromatography video again, now that you have more context