Custom Protein Purification

Immobilized Metal Affinity Chromatography (IMAC)

Immobilized Metal Affinity Chromatography (IMAC) is a chromatographic technique used for the separation and purification of proteins and other biomolecules based on their affinity for metal ions. This method takes advantage of the specific interactions between metal ions and certain amino acid residues, particularly histidine (His), present in the target proteins.

The process typically involves the following steps:

1. Equilibration: The chromatography column is initially equilibrated with a buffer containing a metal-chelating agent, such as nickel or cobalt ions. This creates an environment favorable for the interaction with histidine-tagged proteins.
2. Loading: The protein sample, containing the target protein with the histidine tag, is applied to the column. The histidine residues on the protein form coordinate bonds with the metal ions on the column matrix, leading to the immobilization of the target protein.
3. Washing: Non-specifically bound proteins are washed away with a buffer solution, leaving the histidine-tagged protein of interest attached to the column.
4. Elution: Elution of the purified protein is achieved by introducing a buffer with a higher concentration of imidazole, which competes with the histidine residues for binding to the metal ions. This disrupts the histidine-metal interaction, releasing the target protein into the elution fraction.
5. Analysis: The eluted fractions are typically analyzed using techniques such as SDS-PAGE or Western blotting to assess the purity and molecular weight of the purified protein.

IMAC is particularly useful for purifying recombinant proteins expressed in various host systems, where the addition of a short histidine tag is a common molecular biology technique. The specificity of the histidine-metal interaction allows for a highly selective purification process, resulting in the isolation of the target protein with a high degree of purity.

Affinity chromatography

Affinity chromatography is a powerful and selective technique used in the purification of proteins based on their specific interactions with immobilized ligands. The method exploits the inherent affinity between a target protein and a ligand that is covalently attached to a solid support, such as a chromatography resin. The ligand used is chosen based on its ability to bind selectively to the protein of interest.

Steps in Affinity Chromatography:

1. Equilibration: The chromatography column is first equilibrated with a buffer that promotes binding between the target protein and the immobilized ligand. This establishes a stable environment for the purification process.
2. Sample Application: The protein mixture, containing the protein of interest along with other cellular components, is applied to the column. The target protein selectively binds to the immobilized ligand due to the specific interaction between the ligand and the protein.
3. Washing: Unbound or weakly bound proteins are washed out of the column, leaving only the target protein adhered to the ligand. This step helps remove impurities and ensures a higher purity of the target protein.
4. Elution: The bound target protein is then eluted from the column. This is typically achieved by changing the conditions, such as altering the pH or using a competitive ligand, which disrupts the specific interaction between the protein and the immobilized ligand. Elution results in the release of the purified target protein.
5. Regeneration: The column is regenerated by removing any remaining bound proteins and re-establishing the initial conditions for the next round of purification. This step is crucial for maintaining the column's efficiency and reusability.

Affinity chromatography is highly effective in achieving high purification yields and maintaining the biological activity of the target protein. It is particularly valuable when working with proteins that have specific binding partners or tags, such as His-tagged proteins, enabling a tailored and precise purification process.

Size Exclusion Chromatography (SEC)

Size Exclusion Chromatography (SEC), also known as gel filtration chromatography or gel permeation chromatography, is a chromatographic technique used for the separation and purification of proteins and other biomolecules based on their size. This method is particularly effective for separating molecules in a solution according to their molecular weight.

The process of Size Exclusion Chromatography involves the following steps:

1. Column Packing: The first step is the preparation of a chromatography column filled with a porous stationary phase, usually composed of beads with a defined pore size. Larger molecules cannot penetrate deep into the pores, allowing them to travel through the column more quickly, while smaller molecules enter the pores and take longer to elute.
2. Sample Application: The sample containing a mixture of molecules is applied to the top of the column. As the sample moves down through the column, molecules interact with the stationary phase based on their size.
3. Elution: Elution is the process of the mobile phase (buffer or solvent) passing through the column, carrying the sample components along. Larger molecules, which cannot enter the pores of the stationary phase, travel more quickly through the column and are the first to elute. Smaller molecules enter the pores and take longer to pass through, resulting in a separation based on size.
4. Fraction Collection: As the eluted fractions exit the column, they are collected in separate containers. Each fraction represents molecules of a particular size range.
5. Analysis and Validation: The collected fractions are then analyzed to determine the distribution of sizes within the sample. This analysis may include techniques such as spectrophotometry, gel electrophoresis, or other methods specific to the molecules of interest.

Size Exclusion Chromatography is particularly valuable for separating biomolecules without altering their structure or function, making it a gentle and non-destructive purification technique. It is widely used in biochemistry, molecular biology, and biopharmaceutical research for the purification of proteins, nucleic acids, and other macromolecules based on their size and molecular weight.

Hydrophobic Interaction Chromatography (HIC)

Hydrophobic Interaction Chromatography (HIC) is a chromatographic technique used for the separation and purification of proteins based on their hydrophobicity. Unlike other chromatographic methods that rely on charge or size differences, HIC takes advantage of the hydrophobic regions present on the surfaces of proteins.

The process of Hydrophobic Interaction Chromatography involves the following steps:

1. Column Packing: The chromatography column is filled with a hydrophobic stationary phase, typically a resin or beads coated with hydrophobic groups. The stationary phase interacts with the hydrophobic regions of proteins.
2. Sample Loading: The sample containing the mixture of proteins is applied to the column in a buffer solution that contains a high concentration of a hydrophobic salt, such as ammonium sulfate. This salt competes with the proteins for binding to the hydrophobic groups on the stationary phase.
3. Binding of Proteins: Proteins with hydrophobic regions will interact with the hydrophobic groups on the stationary phase, leading to their temporary immobilization. The strength of binding depends on the degree of hydrophobicity of the proteins.
4. Washing: The column is then washed with a buffer solution containing a decreasing concentration of the hydrophobic salt. This step removes weakly bound or non-specifically bound proteins while retaining those with stronger hydrophobic interactions.
5. Elution: Elution is achieved by decreasing the hydrophobicity of the environment, typically achieved by using a buffer with lower salt concentration or altering the pH. This weakens the interaction between the proteins and the stationary phase, allowing the proteins to be eluted from the column.
6. Fraction Collection: Eluted fractions containing purified proteins are collected separately and can be analyzed for purity and concentration.

HIC is particularly useful for separating proteins with similar charges, making it a valuable technique in the intermediate purification steps of custom protein production. It is often employed in the biopharmaceutical industry for the purification of therapeutic proteins and in research laboratories for various protein purification applications.

Ion Exchange Chromatography (IEX)

Ion Exchange Chromatography (IEX) is a chromatographic technique widely used for the separation and purification of proteins based on their net charge. This method exploits the electrostatic interactions between charged proteins and charged groups on a stationary phase.

The process of Ion Exchange Chromatography involves the following steps:

1. Column Packing: The chromatography column is filled with a stationary phase that contains charged groups. The nature of the charged groups on the stationary phase determines whether it is anion exchange chromatography (AEC) or cation exchange chromatography (CEC). In AEC, the stationary phase has positively charged groups, while in CEC, it has negatively charged groups.
2. Sample Loading: The sample, typically a protein mixture, is applied to the top of the column. Proteins with a net charge opposite to that of the charged groups on the stationary phase will bind to the column.
3. Washing: The column is then washed to remove unbound proteins and other impurities. The washing step helps to enhance the separation by eliminating substances that did not interact specifically with the charged groups.
4. Elution: Elution is achieved by changing the ionic strength of the buffer solution or by adjusting the pH. This disrupts the electrostatic interactions between the proteins and the charged groups on the stationary phase, leading to the release of the bound proteins. Eluted fractions containing the separated proteins are collected.
5. Fraction Collection: The collected fractions are then analyzed for purity and concentration. Techniques such as spectrophotometry or electrophoresis may be employed for this purpose.

Ion Exchange Chromatography is particularly effective for separating proteins that differ in their net charge. It is a valuable technique in the intermediate purification steps of custom protein production and is widely used in biochemistry, biotechnology, and the pharmaceutical industry for the purification of proteins with high specificity. The versatility of ion exchange chromatography makes it suitable for a wide range of applications in the isolation of biomolecules based on their charge characteristics.

Unlocking excellence in custom protein production

At Eurofins CALIXAR, we are committed to being your partner in advancing scientific discovery and innovation. With our custom protein services, we guarantee not only stable, functional, and high-quality proteins but also a seamless journey through the intricate process of protein purification. Our team of experts is dedicated to ensuring the integrity of native full-length proteins, whether they are soluble or membrane-bound, meeting the most stringent quality and purity standards. Elevate your research with confidence by choosing Eurofins CALIXAR for unparalleled excellence in custom protein production and purification.