Gel Electrophoresis

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Gel Electrophoresis
native mobility (voltage)(charge)/(mass) SDS-PA
GE minimizes contribution of charge IEF minimize
s contribution of size

• Isoelectric Focusing
• separates proteins by isoelectric points
• large pore size of gel and equilibrium conditions
  minimize molecular sieving
• native or denaturing conditions possible

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• Carrier Ampholytes
• mixture of aliphatic amines either carboxylic
  or sulfonic acid groups
• generates pH gradient in electric field
• gradient range depends on ampholyte pKa values
• proteins migrate to position isoelectric point

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Preparative IEF (Rotofor?)

• polyester screens separate chamber into 20
  compartments
• fractions rapidly harvested following
  electrofocusing

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IEF Practical Considerations

• gradient range
• low ionic strength for maximum resolution
• gels acetone ppt.
• precipitation problems
• include urea, non-ionic detergents
• heating
• gradient breakdown

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Two-Dimensional Gel Electrophoresis
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Protein Detection Following Electrophoresis

• General Proteins
• organic dyes (eg., Coomassie blue)
• R-250 (slow)
• G-250 (fast)
• silver stain
• 10-100X more sensitive than CB
• fluorescence
• radioactivity
• Specific Proteins
• enzyme activity
• antibody/immunoblot

• Silver Staining Methods
• diamine (ammonical)
• non-diamine
• photodevelopment

• Radiolabeling Proteins
• metabolic
• amino acids
• post-translational
• chemical
• iodination
• alkylating agents

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Autoradiography/Fluorography

• electrophoresis of radioactive proteins
• dry gel and expose to X-ray film
• use intensifying screens for high energy isotopes
• use fluors impregnated in gel for low and medium
  energy isotopes

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Enhancement of Autoradiographic Methods for
Detection of Radioisotopes
Enhancement shortens exposure times by 7-10 fold
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Phosphor Imaging

• filmless autoradiography
• screens contain 'storage-phosphors'
• traps the energy of radioactive emissions
• sensitive to both b-particles and g-rays
• efficiency 100 for particle striking screen
• scanning the screen with a laser beam releases
  the stored energy as light
• fluorescence converted into an image file for
  display and quantification
• high sensitivity ? short exposure times
• range of 5 orders of magnitude
• screens are 'erased' and reused

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Quantifying Proteins

• subjective estimates
• scanning densitometry
• excise bands and count radioactivity

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Protein Detection

• Activity Gels
• carry out electrophoresis under native conditions
• or remove SDS following SDS-PAGE
• some proteins refold
• lower SDS and no heat
• replace with non-ionic detergent

• General Proteins
• Coomassie blue
• silver stains
• fluorescence
• radioactivity
• Specific Proteins
• antibody/immunoblot
• enzyme activity
• protease activity
• redox reactions

• Protease Activity
• co-polymerize PAG with protein substrate
• clear zones following incubation and staining

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Redox Reactions and Tetrazolium Salts

• reduced tetrazolium salts form insoluble formazan
  dyes
• eg, nitro-blue tetrazolium (NBT)
• measure dehydrogenases and other redox reactions
• coupled reactions
• non-redox reactions also possible
• eg, phosphatase (BCIP)