A research question about Biochem

Question

There is a protein that has a pI of 10.5. However, when the protein runs at pH 8 on a reducing SDS-PAGE gel, the protein moves towards the positive electrode. How is this possible?

Answer 1

Hi Quan!

The key to answering your question is to remember that the protein is being analyzed by reducing SDS-PAGE. This technique focuses on the migration of proteins based almost exclusively on their size by eliminating their native charge differences.

Normally, a protein with a pI of 10.5 would be positively charged at pH 8 because pH < pI. However, during SDS-PAGE the protein is denatured and bound by SDS (roughly 1 SDS molecule per 2 amino acid which adds a -1 charge for every 2 amino acids bound) and becomes strongly negatively charged. The total added negative charge is much larger than the original net positive charge of the protein at pH 8. This gives the protein an overall net negative charge which causes the protein to migrate toward the positive electrode.

Hope this helps!

Best,

Josiah T.