Question

In gel electrophoresis, why is it correct to use the term "apparent" in front of molecular weight?

Answer 1

Ans. Every electrophoresis experiment is carried out in specific conditions. The molecular mass of the same protein may vary depending on the conditions of electrophoresis.

Consider the example of BSA - BSA is constituted of a single polypeptide chains with 17 intra-strand disulfide bonds.

I. Presence of reducing agents like b-mercaptoethanol in the reducing buffer cleaves all the disulfide bonds and roughly linearizes it. The protein thus moves through the get depending on its mass.

II. In a non-reducing buffer, all the disulfide bonds remain intact. The protein also retains its 3D structure to a large extent. The protein moves through the gel depending on it mass as well as 3D conformation.

Conclusion: Therefore, due to change in 3D conformation, BSA moves to different distances under reducing and non-reducing conditions causing the difference in its apparent molar mass under the two conditions.

So, the same protein may give different molar mass depending on-

A. The number of polypeptide chains linked by disulfide bonds + reducing agent. All polypeptide chains would be separated and give two or more bands.

B. The number of polypeptide chains linked by disulfide bonds but NO reducing agent. All polypeptide chains remain together and give a single band of relatively higher molar mass than the bands obtained under reducing conditions.

C. Presence of intra-strand disulfide bonds in a single polypeptide chain + reducing agent. All disulfide bonds would be cleaved, the polypeptide chain in linearized and gives a single band by moving through the gel solely bases on its mass.

D. Presence of intra-strand disulfide bonds in a single polypeptide chain but NO reducing agent. All disulfide bonds remains as such, the protein retains most its 3D conformation.

The protein moves through the gel depending on its shape (3D) and mass. The resultant band would be at a different molar mass than the same protein under reducing conditions.

Conclusion: Because none of the values are equal to each other, the molar mass of the protein is specific to different electrophoretic conditions.

So, for each electrophoretic condition, the obtained molar mass of protein is said to be apparent molar mass (that appeared in that specific electrophoretic condition) of the protein in that experiment.