Question

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

Gel-filtration: size; Affinity: specific binding; Ion-exchange: charge; Reverse phase HPLC: hydrophobicity.

Step-by-step solution

- Gel-filtration Chromatography

Gel-filtration chromatography separates proteins based on their size. Larger proteins elute first because they cannot enter the smaller pores in the gel matrix and thus travel through the column faster.

- Affinity Chromatography

Affinity chromatography separates proteins based on their specific interactions with a ligand attached to the chromatography matrix. Only proteins with specific binding affinity to the ligand will be retained on the column, while others will be washed away.

- Ion-Exchange Chromatography

Ion-exchange chromatography separates proteins based on their charge. Proteins are either positively or negatively charged and will bind to oppositely charged resins. Changes in pH or ionic strength can then be used to elute the bound proteins.

- Reverse Phase HPLC

Reverse phase high-performance liquid chromatography (HPLC) separates proteins based on their hydrophobicity. Proteins with more hydrophobic regions will interact more strongly with the hydrophobic stationary phase and elute later compared to less hydrophobic proteins.

Key concepts

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Gel-filtration chromatography separates proteins based on their size. This technique uses a gel matrix with pores of various sizes. As a protein mixture passes through the column, larger proteins cannot enter the smaller pores and thus travel through the column more quickly. In contrast, smaller proteins can enter the pores and take longer to elute. This allows for an effective separation where larger molecules elute first, followed by increasingly smaller ones. It is important to note that gel-filtration chromatography is often also called size-exclusion chromatography. This method is highly useful for analyzing the molecular weight of proteins and for purifying proteins of interest from a mixture.

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Affinity chromatography relies on specific interactions between a protein of interest and a ligand that is attached to the chromatography matrix. The ligand can be any molecule that has a specific binding affinity to a particular protein. When a mixture is passed through the column, only the proteins that bind to the ligand will be retained, while others will be washed away. This type of chromatography is highly selective and can significantly purify a protein from complex mixtures. After binding, the protein can be eluted by changing the conditions (like pH or ionic strength) to disrupt the interaction between the protein and the ligand. This technique is especially useful in the purification of enzymes, antibodies, and other proteins with specific binding characteristics.

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Ion-exchange chromatography relies on the charge of the proteins for separation. This technique uses a resin that has either positive or negative charges. Proteins in the mixture that have an opposite charge to the resin will bind effectively, while those with similar charges will pass through the column. By gradually changing the pH or ionic strength of the elution buffer, the bound proteins are then eluted from the resin. There are two types of ion-exchange chromatography: cation-exchange (where positively charged proteins bind to negatively charged resins) and anion-exchange (where negatively charged proteins bind to positively charged resins). This method is highly effective for isolating proteins based on their isoelectric points and can be used for both purification and analytical purposes.

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Reverse phase high-performance liquid chromatography (HPLC) is a powerful technique for separating proteins based on their hydrophobicity. In this method, the stationary phase is hydrophobic, often consisting of long hydrocarbon chains like C18. As a protein mixture passes through the column, more hydrophobic proteins will have stronger interactions with the stationary phase and consequently elute later than less hydrophobic proteins. The elution is typically achieved by gradually increasing the concentration of an organic solvent in the mobile phase. Reverse phase HPLC provides high resolution and is widely used for analyzing and purifying peptides, proteins, and other biomolecules. It is particularly useful in pharmaceuticals and proteomics for its accuracy and efficiency.