What is gel electrophoresis?

Gel electrophoresis is an analytical technique that separates ions in an electric field and is used extensively in biochemical analysis to separate, identify, and purify proteins, amino acids, and peptides.

In simple terms, different amino acids are put in the middle of a gel at a certain pH. A voltage is applied across the ends of the gel. Amino acids that are positively charged at the particular pH will move towards the negative terminal and those negatively charged will move towards the positive terminal. 

How does an amino acid change when the pH changes?

  • When an amino acid is at the right pH (isoelectric point), it will exist as a zwitterion, which is neutral. (Ref. to image)
  • If the pH is lowered by adding acid, the ─COO− part of the zwitterion accepts an H+ ion to resist the increase in H+ concentration, resulting in a positively charged ion.
  • If the pH is raised by adding a base, the ─NH3+ part of the zwitterion releases an H+ ion to resist the decrease in H+ concentration, resulting in a negatively charged ion.
  • This process also occurs for the side-chains (R group). 
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Example question

  • Three substances are added to the gel: Asp, Lys, and the dipeptide Asp-Lys. The isoelectric point of Asp is 5.4 while the isoelectric point of Lys is 9.8. The gel is at a pH of 5.0.

    Match E, F and G to the three substances.
  • The pH value of the gel is lower than the isoelectric points of all three. Therefore, all exist in a positively charged state as their NH2 groups accept an H+ ion. 
  • G is Lys. Lys is the smallest and has the highest +ve charge. E is Asp, because the amide in the R- group is less able to accept H+ compared to the NH2 group, making it less positive. F is Asp-Lys.
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