Question

RECALL What is the basis for the separation of proteins by the following techniques? (a) gel-filtration chromatography (b) affinity chromatography (c) ion-exchange chromatography (d) reverse phase HPLC

Short answer

(a) size, (b) specific binding interactions, (c) net charge, (d) hydrophobicity

Step-by-step solution

Understand Gel-filtration Chromatography

Gel-filtration chromatography separates proteins based on their size. Larger molecules pass through the column faster because they do not enter the porous beads in the gel matrix, while smaller molecules enter the beads and elute more slowly.

Understand Affinity Chromatography

Affinity chromatography separates proteins based on specific binding interactions between the protein of interest and a ligand that is attached to the chromatography matrix. Proteins that have a high affinity for the ligand will bind and can be eluted later under different conditions to break the interaction.

Understand Ion-exchange Chromatography

Ion-exchange chromatography separates proteins based on their net charge at a given pH. Proteins bind to charged columns with opposite charge (cations bind to cation exchangers and anions bind to anion exchangers) and are eluted by changing the salt concentration or pH of the elution buffer.

Understand Reverse Phase HPLC

Reverse phase High-Performance Liquid Chromatography (HPLC) separates proteins based on their hydrophobicity. Proteins interact with the hydrophobic stationary phase, and less hydrophobic proteins elute first, followed by more hydrophobic proteins as the polarity of the mobile phase decreases.

Key concepts

gel-filtration chromatography

Gel-filtration chromatography, also known as size-exclusion chromatography, is a technique that separates proteins based on their physical size. The column used in this technique is filled with tiny porous beads. When the protein mixture is added to the column, larger proteins cannot enter the pores of the beads. Therefore, they travel through the column quickly.
On the other hand, smaller proteins can enter the pores and take a longer path through the column, making them elute later. This method is especially useful for determining the molecular weight of proteins and separating large molecules from smaller contaminants. It is a gentle method that does not denature the proteins, preserving their functional structures.

affinity chromatography

Affinity chromatography is a powerful technique that exploits specific binding interactions between proteins and specific ligands. The column matrix is coated with a ligand that is specific to the protein of interest.
When the protein mixture is passed through the column, the target protein binds to the ligand, while other proteins flow through. The bound protein can later be eluted by changing the conditions, such as altering pH or salt concentration to disrupt the protein-ligand interaction.
This method is highly specific and effective for purifying proteins from complex mixtures. It is commonly used in the purification of antibodies, enzymes, and hormone receptors.

ion-exchange chromatography

Ion-exchange chromatography separates proteins based on their net charge at a particular pH. The column matrix has charged groups; cation-exchange columns have negatively charged groups that attract positively charged proteins, while anion-exchange columns have positively charged groups that attract negatively charged proteins.
Proteins are bound to the charged groups and are eluted by gradually increasing the salt concentration or adjusting the pH of the buffer. This change in conditions competes with the protein's binding to the matrix, allowing them to be released in order of their charge.
This method is useful in fractionating proteins that have slight differences in their charge properties, allowing for a high degree of resolution.

reverse phase HPLC

Reverse phase High-Performance Liquid Chromatography (HPLC) separates proteins based on hydrophobic interactions. The stationary phase in the column is hydrophobic, usually coated with long-chain hydrocarbons. Proteins in the mixture interact with this hydrophobic stationary phase to varying extents.
Less hydrophobic proteins elute first because they interact less with the stationary phase, while more hydrophobic proteins stay longer in the column. As the polarity of the mobile phase changes, more hydrophobic proteins are gradually eluted.
This technique provides high-resolution separation and is commonly used for analyzing complex protein mixtures, identifying post-translational modifications, and folding variants of proteins.