Gel Electrophoresis

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Gel Electrophoresis
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Steps

• Breakdown the protein into amino acids
• Break amide/peptide bond
• Called hydrolysis (opposite of condensation)
• Acid or base and heat required
• Add protein to gel containing a buffer
• Apply a voltage
• Amino acids move based on mass and charge
• Dye amino acids
• Ninhydrin
• Measure distance traveled,
• Compare to known

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Set-up
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Why amino acids move

• Amino acids separate based on their isoelectric
  point and molar mass
• Isoelectric point
• This is the pH where they net charge of amine and
  carboxylic acid groups cancel out
• NH3-C-COO-

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• pH of buffer is more basic than isoelectric
  point, than amino acid will have a negative
  charge and move toward postive electrode
• There are fewer hydrogen atoms around to
  protonate it
• NH2-C-COO-
• pH of buffer is more acidic than isoelectric
  point, than amino acid will have a positive
  charge and move to negative electrode
• More H atoms around to protonate
• NH3-C-COOH