Download Gel Electrophoresis

Gel Electrophoresis
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
Set-up
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-
• 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