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Electrophoresis Theory

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Electrophoresis Theory Polyacrylamide Gels Basic Apparatus Slab Gels Polyacrylamide Gels Basic Apparatus Slab Gels v = (E/d)(q)/(6 r ) mobility electric field ... – PowerPoint PPT presentation

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Title: Electrophoresis Theory


1
Electrophoresis Theory
2
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3
Gel Electrophoresis
  • friction is ease at which mole-cule passes
    through pores
  • size is the major determinant

mobility ? (voltage)(charge/mass)
4
Polyacrylamide Gels
  • common matrix for gel electrophoresis
  • agarose has larger average pore size

5
Basic Apparatus
  • gel is placed between electrodes
  • buffers complete the circuit
  • proteins loaded onto top of gel

6
Slab Gels
7
Sodium Dodecyl Sulfate (SDS)
  • strongly denaturing detergent
  • disrupts 2o, 3o, and 4o structures
  • binds and confers negative charge to protein
  • charge is proportional to mass

8
SDS-PAGE
  • proteins are unfolded (ie, random coil)
  • uniform charge/mass ratio due to SDS
  • therefore endogenous charge and shape are not
    major factors
  • mobility is inverse of mass

mobility ? (voltage) (charge)
mobility ? (voltage) (mass)
9
Size Standards
  • proteins of known mass used as standards to
    calibrate gels
  • mobility on gels defined as Rf
  • Rf distance protein migrated length of gel
  • or B?B dye front

10
Calculating MW
  • plot log(mass) vs Rf of protein size standards
  • linear
  • extrapolate unknowns
  • relative molecular weight (Mr)
  • some exceptions
  • highly charged proteins
  • some SDS-stable structures

11
Practical Considerations
  • prepare gels
  • choose acrylamide
  • prepare samples
  • stacking gel buffer 2 SDS
  • ? b-mercaptoethanol
  • heating (37o? boil)
  • electrophoresis
  • amount of sample
  • voltage
  • tracking dye (B?B)
  • detect proteins
  • eg, Coomassie blue
  • acrylamide
  • protein size range
  • gradient gels
  • desired resolution
  • amount of sample
  • Voltage
  • ? voltage ? time, but ? heat
  • resistance ? during electrophoresis (EIR)

12
Preparative Electrophoresis
  • high resolution provides analytical information
  • difficult to exploit in protein purification
  • recovery of proteins from gels
  • diffusion
  • electroelution
  • transfer to membrane
  • immunization
  • limited protein capacity
  • special apparatus

13
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