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Electrophoresis - Coggle Diagram

- - - - Cofactor for nucleases
  - - - 'Sinks' to bottom of well
  - - - Carcinogenic
    - - Exited at lower wavelengths
        
        No need to UV light
- - - - Form pores of size similar to molecules
  - - - Avoid post-electrophoretic diffusion
  - - - Holds less water
  - - - Repeating units of
        
        Galactose
        
        3,6-anhydrogalactose
    - - Gel forms when cooled
        
        H bonds form
    - - Conc affects pore size
      - Higher %
        
        Smaller pores
  - - - Using N,N'-methylene bisacrylamide (bis)
      - Polymerisation initiated by free radicals
        
        Produced by ammonium persulphate + TEMED
    - - Paralyses when contact
- - - - I = current
      - R = resistance
      - P = power
  - - - Ohm's law
        
        Resistance falls, current increases
  - - - Long separation times
- - - - Higher charged molecules move faster
    - - Frictional resistance
        
        Small molecules move faster
    - - Linear vs ciruclar DNA
      - Globular proteins vs. fibrous proteins
    - - Electrophoretic mobility (\(\mu\))
        
        Increases with inc. field strength until heating effects occur
- - - - \(pH = pKa + log\frac{[A-]}{AH}\)
      - pH > pKa
        
        Deprotonated state
      - pH < pKa
        
        Protonated state
  - - - Isoelectric point (pI)
    - - Hard to predict pI + pKa
  - - - 37.5:1 standard
    - - Formed from reaction between TEMED + persulphate
    - - 2 gels on top of each other
        
        Proteins all start running gel at same time
      - Stacking gel
        
        Low % acrylamide
        
        Wide pore size
      - Running gel
        
        High % acrylamide
        
        Separation
      - Native PAGE
        
        Mobility is complex
        
        Oligomeric state can play a role
        
        Some proteins never enter gel matrix
        
        Neutral or positively charged
        
        Proteins in natural conformation
      - Dissociating/SDS PAGE
        
        Heat before loading onto gel
        
        In mix of
        
        SDS
        
        Denatures
        
        \(\beta\)-mercaptoethanol
        
        Breaks disulphide bridges
        
        Glycerol
        
        Sink sample into well
        
        Bromophenol blue
        
        Make sample visible
        
        Buffer
        
        Proteins denatured
        
        SDS has charged element
        
        Bigger protein
        
        More SDS
        
        Higher charge
        
        All proteins become charged
        
        Linear chains
        
        Stain protein after
        
        Coomassie blue
        
        ~0.2\(\mu\)g/band for detection
        
        Modern protocols use colloidal form of G-250 stain
        
        2 extra methyl groups
        
        Dye is suspended in aqueous solution - small amount of ethanol
        
        Minimal background staining
        
        Washed with water
        
        e.g. instant blue
        
        Silver staining
        
        Greater sensitivity
        
        Expensive
        
        Laborious
        
        Long protocol
        
        Several hours
    - - %polyacrylamide increases in direction of protein migration
      - Resolve proteins in larger range of Mr
    - - Laser densitometers
      - Gel scanning
      - Digital camera
        
        White glass transilluminator
        
        Band intensity can be quantified by software e.g.
    - - Preserved in 7% acetic acid
      - Dried + stored at room temp.
      - Not necessary
        
        Photo sufficient
    - - Native form
      - pH gradient
        
        Generated by ampholytes
      - When electric field applied
        
        Ampholyte migrates to its own pI
      - Very high resolution
        
        0.01 pH units separation
      - Mix of ampholytes between electrodes