a. You have recently discovered a novel protein having a predicted molecular wei

Question

a. You have recently discovered a novel protein having a predicted molecular weight of 55 kD based on the amino acid sequence and denaturing polyacrylamide gel analysis. After purifying this protein to homogeneity (100% purity), you carry out gel filtration chromatography using a size exclusion column containing Superdex S200 medium and a mobile phase approximating physiological pH and physiological ionic strength (native conditions). After careful comparison with standards (all of which are globular proteins that migrate as expected), you discover that the novel protein, for some reason, elutes with an apparent molecular weight of only 3 kD. Stranger still, when you re-run the eluted protein on another SDS-PAGE gel, you discover that it migrates as a 55 kD protein. What is the most likely explanation for this unusual behavior on the column?

Explanation / Answer

Gel filtration chromatography separates proteins based on molecular weight. Molecules such as peptides and proteins pass through a porous bed and diffuse through the beads in the gel. Smaller proteins will diffuse and move slowly through the gel. However, larger proteins will move faster through the beads and eluted faster. The protein is in its native form.

SDS PAGE is a method that separates proteins based on charge. Sodium dodecyl sulphate will denature the protein, giving the protein a net negative charge. Based on the molecular weight and the amount of negative charge present, the protein is separated. In SDS PAGE, the proteins are separated in denatured state.

There is retardation of the protein in the gel filtration column. One possible explanation is that there is interaction between the protein and the Superdex S200 column. The matrix should be compatible for the separation of protein. Superdex S200 columns are used for globular protein. It is possible that the new protein is not a globular protein and hence, not suitable for the new protein. It may be attaching to the column and hence, eluting later than it should. Reduction of non specific ionic interactions may help in solving this issue. As the protein is showing the right molecular weight on SDS PAGE, the gel filtration is not altering proteins structure.

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