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 +NH3-C-COO- 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