What characteristic of red fluorescent protein is used to separate it by column chromatography?

What characteristic of red fluorescent protein is used to separate it by column chromatography?

• A. The red fluorescent protein has more hydrophobic amino acids than hydrophilic ones, causing it to stick to resin when unfolded.
• B. The red fluorescent protein has a higher molecular weight.
• C. The red fluorescent protein is positively charged.
• D. The red fluorescent protein emits blue fluorescence.

Answer: (A)

Hydrophobic interactions are what proteins often rely on for separation in column chromatography. Red fluorescent protein has a surface with exposed hydrophobic amino acids when it unfolds or is partially denatured, so it tends to bind to a hydrophobic resin more strongly than many other proteins. This binding lets it be retained on the column while more hydrophilic proteins pass through, and it can later be eluted by changing the salt or solvent conditions to disrupt those hydrophobic contacts. The other statements don’t fit the typical separation mechanism: molecular weight would point to size-based separation, a positive charge would imply ion-exchange separation, and blue fluorescence is a property observed after purification rather than a basis for how the sample is separated on the column.

Hydrophobic interactions are what proteins often rely on for separation in column chromatography. Red fluorescent protein has a surface with exposed hydrophobic amino acids when it unfolds or is partially denatured, so it tends to bind to a hydrophobic resin more strongly than many other proteins. This binding lets it be retained on the column while more hydrophilic proteins pass through, and it can later be eluted by changing the salt or solvent conditions to disrupt those hydrophobic contacts. The other statements don’t fit the typical separation mechanism: molecular weight would point to size-based separation, a positive charge would imply ion-exchange separation, and blue fluorescence is a property observed after purification rather than a basis for how the sample is separated on the column.