There are many types of methods for separation and purification of proteins, such as precipitation (specially used salting out, isoelectric precipitation, organic solvent precipitation), membrane separation (ultrafiltration, dialysis), chromatography, centrifugation, etc. . Each type of method has its own characteristics and scope of application. If the crude precipitation method is used for the crude extraction, the precipitation method will not be used for the fine extraction. Among these methods, the most widely used is chromatography.

Chromatography, called chromatography in the biochemical and pharmaceutical industries. However, the Chinese Chemical Society, the relevant departments of the Chinese Academy of Sciences and related journals all require a chromatogram. The term “chromatography” is translated into “chromatography” in national national standards for chromatographic terminology. Therefore, here we separate the column into a chromatogram. The separation medium in the column is also generally referred to as a filler or a stationary phase.

The most important in protein chromatography purification is the choice of separation medium and chromatographic conditions. Some people who work in separation often don't know what to do after getting samples. In fact, the choice of the two can be designed according to the separation object and the separation mechanism of each chromatogram. The choice of protein separation medium should be considered from the molecular structure of the protein.

Compared with the separation and purification of natural products and synthetic compounds of small molecules in general, the separation of proteins has many special features.

1. The structure of a protein molecule determines the choice of method for its separation work.

The protein has a large molecular weight, both above 6KD, and is composed of a plurality of amino acids in a certain sequence through a peptide bond to form a peptide chain, and then one or more peptide chains are combined into a complex three-stage and four-membered by a covalent bond and a non-covalent bond. A protein molecule of a hierarchical structure. Proteins may also bind to sugars, lipids, and nucleic acids to obtain binding proteins, and different binding proteins may differ in properties. The shape of the protein is spherical and fibrous, and its size varies greatly depending on its molecular weight. Therefore, it is possible to separate proteins of different sizes by size exclusion chromatography, ultrafiltration and membrane methods.

Like amino acids, proteins dissociate and form positive or negative ions at different pH. Proteins also have isoelectric points. At the same pH, different proteins are charged differently on the surface, so proteins can be separated by ion exchange chromatography.

The amino acid residues of the protein will have some hydrophobic groups such as phenyl and alkyl groups. These hydrophobic side chains will avoid the aqueous phase in the aqueous phase and be hidden inside the protein molecules to form external hydrophilic groups of the protein molecules. In many cases, the hydrophobic group is mostly inside the protein fissure. Under different conditions, the protein fissure will have different stretching to expose the internal hydrophobic group. Different proteins have different hydrophobicities in the same medium and can be separated by hydrophobic interaction chromatography and reversed phase chromatography.

Biomacromolecules have a property in which certain molecules or groups have a strong specific adsorption. This effect is only for one or a class of target substances. If the antibody specifically adsorbs the antigen, the benzamidine has an adsorption effect on the serine-containing protein. This is the application principle of affinity chromatography. Generally, small molecules do not have such specific adsorption.

2. Pay special attention to maintaining the biological activity of the sample.

Proteins have complex tertiary and quaternary structures. Heat, light, and chemicals cause changes in their physical and chemical properties and loss of physiological activity. This is called inactivation. Inactivation is both reversible and irreversible. Irreversibly inactivated proteins are often solid, insoluble in water and solvents, and no longer have the original physiological properties of the protein. Reversibly inactivated proteins can be restored to their original physiological activities by different renaturation methods.

When selecting and evaluating methods, media and chromatographic conditions for separation and purification, proteins should not only pay attention to the mass recovery rate of the sample, but also pay special attention to the activity recovery rate. To maintain the biological activity of the sample, attention should be paid to the separation method, packing, chromatographic conditions and operating procedures.

In exclusion, ion exchange, hydrophobic and reversed phase, affinity four-class chromatography, reversed-phase chromatography is generally easy to inactivate samples, often used for qualitative, quantitative analysis and amino acid sequence analysis, not for preparation; other chromatographic methods As long as the conditions are properly selected, a high activity recovery rate can be obtained.

3. The chromatographic medium used should be noted for its suitable molecular weight range.

The general medium is porous, and the specific surface of the porous medium is 90% to 95% in the pores. When the separation is made, the inner surface of the hole is fully utilized, and the column capacity can be large. Therefore, a medium having a suitable pore is selected depending on the molecular weight of the sample. The size of the pores in the exclusion chromatography is indicated by the exclusion limit; other chromatograms are expressed by the molecular weight range, which is actually the exclusion limit of the matrix.

Commonly used protein separation media are the agarose and dextran series. The molecular weight of agarose is determined by the concentration of agarose at the time of preparation. The familiar 4B, the concentration of sugar is 4%, suitable for 6 × 10 4 ~ 2 × 10 7 molecular weight; 6B, the concentration of sugar is 6%, for 1 × 10 4 ~ 4 × 10 6 Molecular weight. The separation range of the dextran series depends on the product specification, ranging from a few thousand to tens of millions.

The commonly used silica gel series pore size is 6~12nm for small molecules; 30, 50, 100nm for certain molecular weight proteins.

4. Biomacromolecules have different chromatographic properties than small molecules.

Biomacromolecules, including proteins, have some special chromatographic properties.

In gradient elution chromatography, such as reversed-phase chromatography, hydrophobic chromatography, and ion exchange, the concentration of the strong eluent has a significant jump on the retention value (capacity factor). Organic small molecules do not have this sudden jump. Many problems in protein separation can be explained by this.

5. UV detector detection wavelength is fixed at 280nm and 220nm.

280 nm is the detection of the phenyl group in the protein, and 220 nm is the detection of the peptide bond. Protein detection is often used at 280nm detection wavelength.

Protein separation media selection should consider two aspects at the same time, one is the ligand, which is to choose the chromatographic mode; the other is to select the matrix, including the type of matrix and the choice of the exclusion limit.

Selecting the medium according to the difference in the nature of the target product and impurities is the main basis for medium selection. The media is selected and the split mode is selected.

The difference in the four properties of the target product and impurities in molecular size, isoelectric point pI, hydrophobicity under inactive or reversible inactivation conditions, and interaction with affinity ligands is one of the main basis for medium selection. There is a big difference between the nature of the product and the impurity, which can be used as the basis for the separation method. The difference in molecular size between the target product and the impurity can be separated by gel chromatography; the difference in isoelectric point pI can be separated by ion exchange chromatography; the difference in hydrophobicity under the condition of no inactivation or reversible inactivation can be separated by hydrophobic chromatography; The ligand has a specific affinity and can be separated by affinity chromatography.

In gel chromatography, ion exchange, hydrophobic and reversed phase, affinity chromatography, reversed-phase chromatography is generally easy to inactivate samples, often used for qualitative, quantitative analysis and amino acid sequence analysis, not for preparation; other chromatographic methods As long as the conditions are properly selected, a high activity recovery rate can be obtained, which can be used for preparation separation.

There are three main types of matrix: poly-polysaccharides, such as cross-linked agarose, dextran matrix; silica gel and porous glass; organic polymer (polystyrene, etc.).

Since the target product is limited to proteins and the molecular weight is relatively large, the small pore filler for small molecules cannot be used. Because the total surface area of ​​the porous filler is 90 to 95% in the pores, the spherical surface of the spherical filler only accounts for 5 to 10%. The column capacity of the small pore packing is too small.

Macroporous silica gel and porous glass have poor biocompatibility with proteins. Macroporous silica gel is expensive and has few surface active groups. The prepared filler will retain acidic silicic hydroxyl groups, resulting in non-specific adsorption. Macroporous organic polymers are highly hydrophobic and have poor biocompatibility with proteins, and cannot be used for protein separation without hydrophilic treatment.

Cross-linked agarose and dextran matrix are the most suitable matrix materials for protein separation. These two materials have good biocompatibility, no non-specific adsorption, and a large number of hydroxyl groups on the surface, which can be used to link ligands and produce various kinds of materials. filler.

The agarose microspheres and their crosslinked products are generally called 4B, CL4B, 4FF, 6B, CL6B, and 6FF. These products are then bonded to different groups to obtain ion exchange, hydrophobic, affinity chromatography packing. The 4B, CL4B, and 4FF preparations have a sugar concentration of 4% and an exclusion limit of 60,000 to 20 million. The 6B, CL6B, and 6FF preparations have a sugar concentration of 6% and an exclusion limit of 10,000 to 4 million. 4B and 6B have low withstand voltage; CL4B and CL6B have a slightly higher withstand voltage and are still very low; 4FF and 6FF have higher withstand voltage and can be used up to 0.15 or 0.3MPa.

Glucan is a linear structure that is crosslinked into spheres with epichlorohydrin. Due to the difference in sugar concentration and cross-linking degree, microspheres with different exclusion limits were obtained. The exclusion range of dextran microspheres is smaller, and the column volume will change greatly under different conditions. It has been used less frequently for ion exchange, hydrophobic, affinity chromatography, and more for gel chromatography.

Xi'an Hengcheng Biotechnology Co., Ltd. is a company specializing in the research, production and marketing of chromatography media (chromatography packing);

There are two major categories of products: unliganded microspheres and chromatographic media

The five major chromatographic media are: ion exchange, exclusion (gel filtration), affinity, hydrophobic, reverse;

Three types of materials: agarose gel, dextran gel, silica gel

Ion exchange includes: cation (SP, CM) anion (Q, DEAE);

Gel filtration includes: 4B, 6B, CL-4B, CL-6B, 4BFF, 6BFF

Hydrophobicity includes: phenyl, butyl

Affinity: protein a column, endotoxin column, heparin column, gelatin column, lysine column, boric acid column, ConA column, nickel column and so on.

Affinity activation intermediates are: bromination * activation 4B, amino-based 4B, carboxy-based 4B, epoxy activated FF, NHS activation, etc.;

There are C18, C8, C12 in the reverse. Source: There are many kinds of methods for separation and purification of proteins, such as precipitation method (especially common salting out method, isoelectric precipitation method, organic solvent precipitation method), membrane separation method (ultrafiltration method, Dialysis method, chromatography, centrifugation, etc. Each type of method has its own characteristics and scope of application. If the crude precipitation method is used for the crude extraction, the precipitation method will not be used for the fine extraction. Among these methods, the most widely used is chromatography.

Chromatography, called chromatography in the biochemical and pharmaceutical industries. However, the Chinese Chemical Society, the relevant departments of the Chinese Academy of Sciences and related journals all require a chromatogram. The term “chromatography” is translated into “chromatography” in national national standards for chromatographic terminology. Therefore, here we separate the column into a chromatogram. The separation medium in the column is also generally referred to as a filler or a stationary phase.

The most important in protein chromatography purification is the choice of separation medium and chromatographic conditions. Some people who work in separation often don't know what to do after getting samples. In fact, the choice of the two can be designed according to the separation object and the separation mechanism of each chromatogram. The choice of protein separation medium should be considered from the molecular structure of the protein.

Compared with the separation and purification of natural products and synthetic compounds of small molecules in general, the separation of proteins has many special features.

1. The structure of a protein molecule determines the choice of method for its separation work.

The protein has a large molecular weight, both above 6KD, and is composed of a plurality of amino acids in a certain sequence through a peptide bond to form a peptide chain, and then one or more peptide chains are combined into a complex three-stage and four-membered by a covalent bond and a non-covalent bond. A protein molecule of a hierarchical structure. Proteins may also bind to sugars, lipids, and nucleic acids to obtain binding proteins, and different binding proteins may differ in properties. The shape of the protein is spherical and fibrous, and its size varies greatly depending on its molecular weight. Therefore, it is possible to separate proteins of different sizes by size exclusion chromatography, ultrafiltration and membrane methods.

Like amino acids, proteins dissociate and form positive or negative ions at different pH. Proteins also have isoelectric points. At the same pH, different proteins are charged differently on the surface, so proteins can be separated by ion exchange chromatography.

The amino acid residues of the protein will have some hydrophobic groups such as phenyl and alkyl groups. These hydrophobic side chains will avoid the aqueous phase in the aqueous phase and be hidden inside the protein molecules to form external hydrophilic groups of the protein molecules. In many cases, the hydrophobic group is mostly inside the protein fissure. Under different conditions, the protein fissure will have different stretching to expose the internal hydrophobic group. Different proteins have different hydrophobicities in the same medium and can be separated by hydrophobic interaction chromatography and reversed phase chromatography.

Biomacromolecules have a property in which certain molecules or groups have a strong specific adsorption. This effect is only for one or a class of target substances. If the antibody specifically adsorbs the antigen, the benzamidine has an adsorption effect on the serine-containing protein. This is the application principle of affinity chromatography. Generally, small molecules do not have such specific adsorption.

2. Pay special attention to maintaining the biological activity of the sample.

Proteins have complex tertiary and quaternary structures. Heat, light, and chemicals cause changes in their physical and chemical properties and loss of physiological activity. This is called inactivation. Inactivation is both reversible and irreversible. Irreversibly inactivated proteins are often solid, insoluble in water and solvents, and no longer have the original physiological properties of the protein. Reversibly inactivated proteins can be restored to their original physiological activities by different renaturation methods.

When selecting and evaluating methods, media and chromatographic conditions for separation and purification, proteins should not only pay attention to the mass recovery rate of the sample, but also pay special attention to the activity recovery rate. To maintain the biological activity of the sample, attention should be paid to the separation method, packing, chromatographic conditions and operating procedures.

In exclusion, ion exchange, hydrophobic and reversed phase, affinity four-class chromatography, reversed-phase chromatography is generally easy to inactivate samples, often used for qualitative, quantitative analysis and amino acid sequence analysis, not for preparation; other chromatographic methods As long as the conditions are properly selected, a high activity recovery rate can be obtained.

3. The chromatographic medium used should be noted for its suitable molecular weight range.

The general medium is porous, and the specific surface of the porous medium is 90% to 95% in the pores. When the separation is made, the inner surface of the hole is fully utilized, and the column capacity can be large. Therefore, a medium having a suitable pore is selected depending on the molecular weight of the sample. The size of the pores in the exclusion chromatography is indicated by the exclusion limit; other chromatograms are expressed by the molecular weight range, which is actually the exclusion limit of the matrix.

Commonly used protein separation media are the agarose and dextran series. The molecular weight of agarose is determined by the concentration of agarose at the time of preparation. The familiar 4B, the concentration of sugar is 4%, suitable for 6 × 10 4 ~ 2 × 10 7 molecular weight; 6B, the concentration of sugar is 6%, for 1 × 10 4 ~ 4 × 10 6 Molecular weight. The separation range of the dextran series depends on the product specification, ranging from a few thousand to tens of millions.

The commonly used silica gel series pore size is 6~12nm for small molecules; 30, 50, 100nm for certain molecular weight proteins.

4. Biomacromolecules have different chromatographic properties than small molecules.

Biomacromolecules, including proteins, have some special chromatographic properties.

In gradient elution chromatography, such as reversed-phase chromatography, hydrophobic chromatography, and ion exchange, the concentration of the strong eluent has a significant jump on the retention value (capacity factor). Organic small molecules do not have this sudden jump. Many problems in protein separation can be explained by this.

5. UV detector detection wavelength is fixed at 280nm and 220nm.

280 nm is the detection of the phenyl group in the protein, and 220 nm is the detection of the peptide bond. Protein detection is often used at 280nm detection wavelength.

Protein separation media selection should consider two aspects at the same time, one is the ligand, which is to choose the chromatographic mode; the other is to select the matrix, including the type of matrix and the choice of the exclusion limit.

Selecting the medium according to the difference in the nature of the target product and impurities is the main basis for medium selection. The media is selected and the split mode is selected.

The difference in the four properties of the target product and impurities in molecular size, isoelectric point pI, hydrophobicity under inactive or reversible inactivation conditions, and interaction with affinity ligands is one of the main basis for medium selection. There is a big difference between the nature of the product and the impurity, which can be used as the basis for the separation method. The difference in molecular size between the target product and the impurity can be separated by gel chromatography; the difference in isoelectric point pI can be separated by ion exchange chromatography; the difference in hydrophobicity under the condition of no inactivation or reversible inactivation can be separated by hydrophobic chromatography; The ligand has a specific affinity and can be separated by affinity chromatography.

In gel chromatography, ion exchange, hydrophobic and reversed phase, affinity chromatography, reversed-phase chromatography is generally easy to inactivate samples, often used for qualitative, quantitative analysis and amino acid sequence analysis, not for preparation; other chromatographic methods As long as the conditions are properly selected, a high activity recovery rate can be obtained, which can be used for preparation separation.

There are three main types of matrix: poly-polysaccharides, such as cross-linked agarose, dextran matrix; silica gel and porous glass; organic polymer (polystyrene, etc.).

Since the target product is limited to proteins and the molecular weight is relatively large, the small pore filler for small molecules cannot be used. Because the total surface area of ​​the porous filler is 90 to 95% in the pores, the spherical surface of the spherical filler only accounts for 5 to 10%. The column capacity of the small pore packing is too small.

Macroporous silica gel and porous glass have poor biocompatibility with proteins. Macroporous silica gel is expensive and has few surface active groups. The prepared filler will retain acidic silicic hydroxyl groups, resulting in non-specific adsorption. Macroporous organic polymers are highly hydrophobic and have poor biocompatibility with proteins, and cannot be used for protein separation without hydrophilic treatment.

Cross-linked agarose and dextran matrix are the most suitable matrix materials for protein separation. These two materials have good biocompatibility, no non-specific adsorption, and a large number of hydroxyl groups on the surface, which can be used to link ligands and produce various kinds of materials. filler.

The agarose microspheres and their crosslinked products are generally called 4B, CL4B, 4FF, 6B, CL6B, and 6FF. These products are then bonded to different groups to obtain ion exchange, hydrophobic, affinity chromatography packing. The 4B, CL4B, and 4FF preparations have a sugar concentration of 4% and an exclusion limit of 60,000 to 20 million. The 6B, CL6B, and 6FF preparations have a sugar concentration of 6% and an exclusion limit of 10,000 to 4 million. 4B and 6B have low withstand voltage; CL4B and CL6B have a slightly higher withstand voltage and are still very low; 4FF and 6FF have higher withstand voltage and can be used up to 0.15 or 0.3MPa.

Glucan is a linear structure that is crosslinked into spheres with epichlorohydrin. Due to the difference in sugar concentration and cross-linking degree, microspheres with different exclusion limits were obtained. The exclusion range of dextran microspheres is smaller, and the column volume will change greatly under different conditions. It has been used less frequently for ion exchange, hydrophobic, affinity chromatography, and more for gel chromatography.

Xi'an Hengcheng Biotechnology Co., Ltd. is a company specializing in the research, production and marketing of chromatography media (chromatography packing);

There are two major categories of products: unliganded microspheres and chromatographic media

The five major chromatographic media are: ion exchange, exclusion (gel filtration), affinity, hydrophobic, reverse;

Three types of materials: agarose gel, dextran gel, silica gel

Ion exchange includes: cation (SP, CM) anion (Q, DEAE);

Gel filtration includes: 4B, 6B, CL-4B, CL-6B, 4BFF, 6BFF

Hydrophobicity includes: phenyl, butyl

Affinity: protein a column, endotoxin column, heparin column, gelatin column, lysine column, boric acid column, ConA column, nickel column and so on.

Affinity activation intermediates are: bromination * activation 4B, amino-based 4B, carboxy-based 4B, epoxy activated FF, NHS activation, etc.;

There are C18, C8, C12 sources in the reverse direction: there are many kinds of methods for separation and purification of proteins, such as precipitation method (specially used salting out method, isoelectric precipitation method, organic solvent precipitation method), membrane separation method (ultrafiltration method, Dialysis method, chromatography, centrifugation, etc. Each type of method has its own characteristics and scope of application. If the crude precipitation method is used for the crude extraction, the precipitation method will not be used for the fine extraction. Among these methods, the most widely used is chromatography.

Chromatography, called chromatography in the biochemical and pharmaceutical industries. However, the Chinese Chemical Society, the relevant departments of the Chinese Academy of Sciences and related journals all require a chromatogram. The term “chromatography” is translated into “chromatography” in national national standards for chromatographic terminology. Therefore, here we separate the column into a chromatogram. The separation medium in the column is also generally referred to as a filler or a stationary phase.

The most important in protein chromatography purification is the choice of separation medium and chromatographic conditions. Some people who work in separation often don't know what to do after getting samples. In fact, the choice of the two can be designed according to the separation object and the separation mechanism of each chromatogram. The choice of protein separation medium should be considered from the molecular structure of the protein.

Compared with the separation and purification of natural products and synthetic compounds of small molecules in general, the separation of proteins has many special features.

1. The structure of a protein molecule determines the choice of method for its separation work.

The protein has a large molecular weight, both above 6KD, and is composed of a plurality of amino acids in a certain sequence through a peptide bond to form a peptide chain, and then one or more peptide chains are combined into a complex three-stage and four-membered by a covalent bond and a non-covalent bond. A protein molecule of a hierarchical structure. Proteins may also bind to sugars, lipids, and nucleic acids to obtain binding proteins, and different binding proteins may differ in properties. The shape of the protein is spherical and fibrous, and its size varies greatly depending on its molecular weight. Therefore, it is possible to separate proteins of different sizes by size exclusion chromatography, ultrafiltration and membrane methods.

Like amino acids, proteins dissociate and form positive or negative ions at different pH. Proteins also have isoelectric points. At the same pH, different proteins are charged differently on the surface, so proteins can be separated by ion exchange chromatography.

The amino acid residues of the protein will have some hydrophobic groups such as phenyl and alkyl groups. These hydrophobic side chains will avoid the aqueous phase in the aqueous phase and be hidden inside the protein molecules to form external hydrophilic groups of the protein molecules. In many cases, the hydrophobic group is mostly inside the protein fissure. Under different conditions, the protein fissure will have different stretching to expose the internal hydrophobic group. Different proteins have different hydrophobicities in the same medium and can be separated by hydrophobic interaction chromatography and reversed phase chromatography.

Biomacromolecules have a property in which certain molecules or groups have a strong specific adsorption. This effect is only for one or a class of target substances. If the antibody specifically adsorbs the antigen, the benzamidine has an adsorption effect on the serine-containing protein. This is the application principle of affinity chromatography. Generally, small molecules do not have such specific adsorption.

2. Pay special attention to maintaining the biological activity of the sample.

Proteins have complex tertiary and quaternary structures. Heat, light, and chemicals cause changes in their physical and chemical properties and loss of physiological activity. This is called inactivation. Inactivation is both reversible and irreversible. Irreversibly inactivated proteins are often solid, insoluble in water and solvents, and no longer have the original physiological properties of the protein. Reversibly inactivated proteins can be restored to their original physiological activities by different renaturation methods.

When selecting and evaluating methods, media and chromatographic conditions for separation and purification, proteins should not only pay attention to the mass recovery rate of the sample, but also pay special attention to the activity recovery rate. To maintain the biological activity of the sample, attention should be paid to the separation method, packing, chromatographic conditions and operating procedures.

In exclusion, ion exchange, hydrophobic and reversed phase, affinity four-class chromatography, reversed-phase chromatography is generally easy to inactivate samples, often used for qualitative, quantitative analysis and amino acid sequence analysis, not for preparation; other chromatographic methods As long as the conditions are properly selected, a high activity recovery rate can be obtained.

3. The chromatographic medium used should be noted for its suitable molecular weight range.

The general medium is porous, and the specific surface of the porous medium is 90% to 95% in the pores. When the separation is made, the inner surface of the hole is fully utilized, and the column capacity can be large. Therefore, a medium having a suitable pore is selected depending on the molecular weight of the sample. The size of the pores in the exclusion chromatography is indicated by the exclusion limit; other chromatograms are expressed by the molecular weight range, which is actually the exclusion limit of the matrix.

Commonly used protein separation media are the agarose and dextran series. The molecular weight of agarose is determined by the concentration of agarose at the time of preparation. The familiar 4B, the concentration of sugar is 4%, suitable for 6 × 10 4 ~ 2 × 10 7 molecular weight; 6B, the concentration of sugar is 6%, for 1 × 10 4 ~ 4 × 10 6 Molecular weight. The separation range of the dextran series depends on the product specification, ranging from a few thousand to tens of millions.

The commonly used silica gel series pore size is 6~12nm for small molecules; 30, 50, 100nm for certain molecular weight proteins.

4. Biomacromolecules have different chromatographic properties than small molecules.

Biomacromolecules, including proteins, have some special chromatographic properties.

In gradient elution chromatography, such as reversed-phase chromatography, hydrophobic chromatography, and ion exchange, the concentration of the strong eluent has a significant jump on the retention value (capacity factor). Organic small molecules do not have this sudden jump. Many problems in protein separation can be explained by this.

5. UV detector detection wavelength is fixed at 280nm and 220nm.

280 nm is the detection of the phenyl group in the protein, and 220 nm is the detection of the peptide bond. Protein detection is often used at 280nm detection wavelength.

Protein separation media selection should consider two aspects at the same time, one is the ligand, which is to choose the chromatographic mode; the other is to select the matrix, including the type of matrix and the choice of the exclusion limit.

Selecting the medium according to the difference in the nature of the target product and impurities is the main basis for medium selection. The media is selected and the split mode is selected.

The difference in the four properties of the target product and impurities in molecular size, isoelectric point pI, hydrophobicity under inactive or reversible inactivation conditions, and interaction with affinity ligands is one of the main basis for medium selection. There is a big difference between the nature of the product and the impurity, which can be used as the basis for the separation method. The difference in molecular size between the target product and the impurity can be separated by gel chromatography; the difference in isoelectric point pI can be separated by ion exchange chromatography; the difference in hydrophobicity under the condition of no inactivation or reversible inactivation can be separated by hydrophobic chromatography; The ligand has a specific affinity and can be separated by affinity chromatography.

In gel chromatography, ion exchange, hydrophobic and reversed phase, affinity chromatography, reversed-phase chromatography is generally easy to inactivate samples, often used for qualitative, quantitative analysis and amino acid sequence analysis, not for preparation; other chromatographic methods As long as the conditions are properly selected, a high activity recovery rate can be obtained, which can be used for preparation separation.

There are three main types of matrix: poly-polysaccharides, such as cross-linked agarose, dextran matrix; silica gel and porous glass; organic polymer (polystyrene, etc.).

Since the target product is limited to proteins and the molecular weight is relatively large, the small pore filler for small molecules cannot be used. Because the total surface area of ​​the porous filler is 90 to 95% in the pores, the spherical surface of the spherical filler only accounts for 5 to 10%. The column capacity of the small pore packing is too small.

Macroporous silica gel and porous glass have poor biocompatibility with proteins. Macroporous silica gel is expensive and has few surface active groups. The prepared filler will retain acidic silicic hydroxyl groups, resulting in non-specific adsorption. Macroporous organic polymers are highly hydrophobic and have poor biocompatibility with proteins, and cannot be used for protein separation without hydrophilic treatment.

Cross-linked agarose and dextran matrix are the most suitable matrix materials for protein separation. These two materials have good biocompatibility, no non-specific adsorption, and a large number of hydroxyl groups on the surface, which can be used to link ligands and produce various kinds of materials. filler.

The agarose microspheres and their crosslinked products are generally called 4B, CL4B, 4FF, 6B, CL6B, and 6FF. These products are then bonded to different groups to obtain ion exchange, hydrophobic, affinity chromatography packing. The 4B, CL4B, and 4FF preparations have a sugar concentration of 4% and an exclusion limit of 60,000 to 20 million. The 6B, CL6B, and 6FF preparations have a sugar concentration of 6% and an exclusion limit of 10,000 to 4 million. 4B and 6B have low withstand voltage; CL4B and CL6B have a slightly higher withstand voltage and are still very low; 4FF and 6FF have higher withstand voltage and can be used up to 0.15 or 0.3MPa.

Glucan is a linear structure that is crosslinked into spheres with epichlorohydrin. Due to the difference in sugar concentration and cross-linking degree, microspheres with different exclusion limits were obtained. The exclusion range of dextran microspheres is smaller, and the column volume will change greatly under different conditions. It has been used less frequently for ion exchange, hydrophobic, affinity chromatography, and more for gel chromatography.

Xi'an Hengcheng Biotechnology Co., Ltd. is a company specializing in the research, production and marketing of chromatography media (chromatography packing);

There are two major categories of products: unliganded microspheres and chromatographic media

The five major chromatographic media are: ion exchange, exclusion (gel filtration), affinity, hydrophobic, reverse;

Three types of materials: agarose gel, dextran gel, silica gel

Ion exchange includes: cation (SP, CM) anion (Q, DEAE);

Gel filtration includes: 4B, 6B, CL-4B, CL-6B, 4BFF, 6BFF

Hydrophobicity includes: phenyl, butyl

Affinity: protein a column, endotoxin column, heparin column, gelatin column, lysine column, boric acid column, ConA column, nickel column and so on.

Affinity activation intermediates are: bromination * activation 4B, amino-based 4B, carboxy-based 4B, epoxy activated FF, NHS activation, etc.;

Reversed are C18, C8, C12

Source: Xi'an Hengcheng Biotechnology Co., Ltd.

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