Boronate Affinity Chromatography Resin (C008B-270281)

This product is an affinity chromatography medium formed by designing a specific spacer arm on the surface of a highly rigid agarose gel microsphere matrix, followed by coupling with 3-aminophenylboronic acid. Under weakly alkaline conditions, the phenylboronic acid ligand can form a boronate ester with 1,2-cis-diol through a reversible covalent bond, enabling specific binding to substances containing 1,2-cis-diol structures, including glycoproteins (including antibodies), enzymes, polysaccharides, nucleosides, nucleotides, catecholamines and other biomolecules. It can also assist in the separation and purification of target molecules that cannot directly bind to the resin with NADP⁺ or ATP. Due to its high specificity and wide application, the purification process using this product can be independently classified as Boronate Affinity Chromatography (BAC).Aladdin  Boronate Affinity Chromatography Resin is stored in 20% ethanol, with a volume ratio of gel to protective solution of 1:1. The product specification refers to the actual gel volume.ItemDetailsMatrixHighly rigid agaroseLigand3-aminophenylboronic acid, ~20 μmol/mLParticle size range① 30–100 μmMean particle size~60 μmDynamic binding capacity② ~10 mg h-IgG/mLRecommended operating flow rate30–150 cm/hMaximum flow rate and pressure③ 1500 cm/h, 0.5 MPaWorking pH3–10 (operation); 2–13 (CIP process)Chemical stabilityStable in common aqueous buffers, 0.5 mol/L NaOH, 8 mol/L urea, 30% isopropanol, 70% ethanol, etc.Storage20% ethanol, 2–8 °CShelf life3 yearsNotes:①More than 90% of the microspheres are within this particle size range.② Test method: 2-hour incubation; the result is close to DBC₁₀%.③ Maximum tested flow rate at a column height of 10 cm.Principle of Boronate AffinityThe main interaction during chromatography is the reversible covalent bond (boronate ester) formed between the boronate affinity chromatography medium and 1,2-cis-diol structures (Figure 1). A pH above 8.5 favors the binding of the resin to the target molecule in the form of boronate ester, while conditions above pH 9.0 are rarely used for the separation and purification of biomolecules; a pH of 4.0–6.0 favors the dissociation of boronate ester. In addition to eluting the target molecule by lowering the pH, competitive elution can also be performed with sugars containing 1,2-cis-diol structures such as sorbitol and mannitol.In addition to reversible covalent bonds, the ligand contains a benzene ring as a hydrophobic group, which may have π–π interactions with benzene ring-containing substances. This requires that the ionic strength of the loading buffer should not be too high, usually about 50 mM, to reduce non-specific adsorption caused by hydrophobic interactions. The tetrahedral structure of boronate ester is negatively charged; to reduce non-specific adsorption caused by ionic interactions, a high ionic strength is required during the binding process. Boronate affinity chromatography medium usually achieves optimal specific binding to target molecules at an ionic strength of 50–500 mM. Divalent cations can reduce non-specific adsorption caused by ionic and hydrophobic interactions; for example, 0–50 mM MgCl sub>2 can be added to the loading buffer. Mg2+ can also inhibit charge repulsion between the resin and phosphate-containing substances such as nucleic acids, and adding Mg2+ can enhance the binding effect.When the formed boronate ester exists as an uncharged planar triangular structure, the boron atom has an empty orbital and can act as an electron acceptor for charge-transfer interactions. Unprotonated amino groups are good electron donors; when the amino group provides a lone pair of electrons to the boron atom, the boron atom forms a tetrahedral shape, promoting the formation of boronate ester. However, it should be noted that when a hydroxyl group exists near the amino group, the resin cannot form a boronate ester structure with 1,2-cis-diol. This is why ethanolamine and Tris are not suitable as loading buffers.Figure 1. Affinity principle of boronate affinity chromatography mediumInstructions for Use1. Column PackingThe column packing method for the resin connected to the chromatography system is described below.(1)Equilibrate all materials to the same temperature as the chromatography operation; liquids are preferably degassed.(2)Resin dosage calculation: Quantify the resin volume by sedimentation. Required settled resin volume = Column volume × Compression factor. The settled volume is the stable volume read after complete natural sedimentation of the resin in 20% ethanol storage solution. The compression factor of boronate affinity chromatography medium is ~1.10. To achieve the compression factor, compression can be performed by lowering the column top or at a high flow rate.(3)Resin washing: Mix the resin suspension thoroughly, measure the corresponding volume, remove the liquid, and wash with approximately 3 resin volumes of purified water, repeated 3 times to remove the storage solution.(4)Packing slurry preparation: Transfer the resin to a suitable container, add an appropriate amount of packing buffer to prepare a 50–75 v% packing slurry (the original package contains 50% resin by volume), and mix well before use.(5)Pre-packing preparation: Add packing buffer to the bottom of the cleaned chromatography column to remove air from the lower frit and the column bottom, leave a small amount of distilled water in the column, tighten the lower plug, and adjust the column to be vertical to the ground.(6)Packing: Pour the well-mixed resin suspension slowly into the chromatography column at one time (use a packing device if necessary). To avoid introducing air bubbles, let it flow naturally along the inner wall of the chromatography column. After adding all the resin, fill the packing device with packing buffer, tighten the top cover of the packing device, and connect the column to the chromatography system.(7)Compaction: Let the resin settle naturally (or at a low flow rate of 50–150 cm/h). After complete sedimentation (clear interface between resin and liquid), remove the packing device, install the upper column top, and lower the top to the interface; continue compaction at a high flow rate (see the table below for recommended flow rate; note that column pressure does not exceed 0.3 MPa) until the interface is clear and stable, and mark the column height when the interface is stable. Stop the pump, open the valve/plug on the column top, close the valve/plug at the column bottom, lower the top to the position below the marked position corresponding to the compression factor, tighten the top, and column packing is completed. After packing, equilibrate the resin in the column at a high flow rate.Packing ConditionsBoronate Affinity Chromatography ResinCompression factor~1.10Packing flow rate600–1500 cm/h, depending on column size, subject to compression factor and column efficiency test2. Column Efficiency Determination and EvaluationColumn efficiency can be measured and evaluated to confirm the packing quality after packing and before use. Column efficiency is usually evaluated by Height Equivalent to a Theoretical Plate (HETP) and Asymmetry factor (As).Column efficiency can be determined using acetone or NaCl as samples. Prepare sample solutions and mobile phases according to the table below.Item1.0% Acetone0.8–1.0 M NaClSample volume1.0% column volume1.0% column volumeMobile phasePure water0.4 M NaClFlow rate30 cm/h30 cm/hDetectorUV-280 nmConductivityCalculate HETP, Number of theoretical plates (N) and As based on UV or conductivity curves using the following formulas:HETP = L / NN = 5.54 × (Vr / Wh)2As = a / bWhere:L = column height; Vr = retention volume; Wh = peak width at half height; a = first half-peak width at 10% peak height; b = second half-peak width at 10% peak height.Generally, HETP should be less than three times the mean particle size of the resin (i.e., HETP/D50 < 3, D50 = mean particle size of the resin), and As should be between 0.8 and 1.5.3. Equilibration and Sample LoadingBefore sample loading, equilibrate the column with loading buffer (equilibration buffer) at the operating flow rate, and observe changes in the detector until parameters such as conductivity and pH remain constant. The sample loading volume is selected according to the sample properties and the amount of chromatography medium, which can be determined in preliminary experiments (e.g., static adsorption experiment). Generally, the sample needs to be diluted with loading buffer, e.g., 1:1 dilution.The pH of the loading buffer is usually 8.5–9.0. For example, HEPES buffer (20 mM HEPES, 20 mM MgCl2, pH 8.5) can be used to separate glycoproteins from non-glycoproteins, and ammonium acetate buffer (0.25 M ammonium acetate, pH 8.8) can be used to separate ribonucleosides from deoxyribonucleosides. Common loading buffer systems for this resin include phosphate, ammonium acetate, HEPES, etc. In general, amino-containing buffers such as Tris-HCl should be avoided.4. ElutionWash the loaded column with 2–3 column volumes of equilibration buffer, and observe changes in the detector until parameters such as conductivity and pH remain constant. At this time, unbound components are washed out.Elution of affinity chromatography columns can be performed by isocratic elution, gradient elution or step elution. During elution, the pH can be reduced to 4–6 (e.g., 0.1 M formic acid, 25 mM hydrochloric acid); sugars can also be selected for competitive elution, such as 10–200 mM sorbitol; 0–20 mM EDTA can also be used; Tris can be used as a high-efficiency eluent.5. Regeneration and Cleaning-in-Place (CIP)The resin can usually be regenerated with 5 CV of eluent. If inactivated proteins or lipids cannot be washed out during regeneration, they can be removed by Cleaning-in-Place (CIP). The following options can be used for CIP: 0.5 mol/L NaOH, 70% ethanol, 30% isopropanol, etc. CIP can effectively remove impurities on the medium, and the effect is better in reverse flow. Refer to the table below for specific CIP solution selection.CIP ProcessPurpose0.5 mol/L NaOH (minimize cleaning time); water, 5 CVRemove protein precipitates or other impurities such as pyrogens30% isopropanol or 70% ethanol, 4–10 CV, <50 cm/h; water, 5 CVRemove tightly bound lipoproteins, liposomes, etc.6. Resin StorageThe initial storage solution of the resin is 20% ethanol, and the resin can continue to be stored in 20% ethanol after use. It can also be stored in buffers similar to elution and regeneration, such as 0.2 M sodium acetate and 0.1 M formic acid. 0.02% sodium azide can be added to the buffer for antisepsis. The resin is more stable in a weakly acidic environment. The storage temperature is preferably 2–8 °C; do not freeze.

Specifications & Purity: This product is an affinity chromatography medium formed by designing a specific spacer arm on the surface of a highly rigid agarose gel microsphere matrix, followed by coupling with 3-aminophenylboronic acid. Under weakly alkaline conditions, the phenylboronic acid ligand can form a boronate ester with 1,2-cis-diol through a reversible covalent bond, enabling specific binding to substances containing 1,2-cis-diol structures, including glycoproteins (including antibodies), enzymes, polysaccharides, nucleosides, nucleotides, catecholamines and other biomolecules. It can also assist in the separation and purification of target molecules that cannot directly bind to the resin with NADP⁺ or ATP. Due to its high specificity and wide application, the purification process using this product can be independently classified as Boronate Affinity Chromatography (BAC).Aladdin  Boronate Affinity Chromatography Resin is stored in 20% ethanol, with a volume ratio of gel to protective solution of 1:1. The product specification refers to the actual gel volume.ItemDetailsMatrixHighly rigid agaroseLigand3-aminophenylboronic acid, ~20 μmol/mLParticle size range① 30–100 μmMean particle size~60 μmDynamic binding capacity② ~10 mg h-IgG/mLRecommended operating flow rate30–150 cm/hMaximum flow rate and pressure③ 1500 cm/h, 0.5 MPaWorking pH3–10 (operation); 2–13 (CIP process)Chemical stabilityStable in common aqueous buffers, 0.5 mol/L NaOH, 8 mol/L urea, 30% isopropanol, 70% ethanol, etc.Storage20% ethanol, 2–8 °CShelf life3 yearsNotes:①More than 90% of the microspheres are within this particle size range.② Test method: 2-hour incubation; the result is close to DBC₁₀%.③ Maximum tested flow rate at a column height of 10 cm.Principle of Boronate AffinityThe main interaction during chromatography is the reversible covalent bond (boronate ester) formed between the boronate affinity chromatography medium and 1,2-cis-diol structures (Figure 1). A pH above 8.5 favors the binding of the resin to the target molecule in the form of boronate ester, while conditions above pH 9.0 are rarely used for the separation and purification of biomolecules; a pH of 4.0–6.0 favors the dissociation of boronate ester. In addition to eluting the target molecule by lowering the pH, competitive elution can also be performed with sugars containing 1,2-cis-diol structures such as sorbitol and mannitol.In addition to reversible covalent bonds, the ligand contains a benzene ring as a hydrophobic group, which may have π–π interactions with benzene ring-containing substances. This requires that the ionic strength of the loading buffer should not be too high, usually about 50 mM, to reduce non-specific adsorption caused by hydrophobic interactions. The tetrahedral structure of boronate ester is negatively charged; to reduce non-specific adsorption caused by ionic interactions, a high ionic strength is required during the binding process. Boronate affinity chromatography medium usually achieves optimal specific binding to target molecules at an ionic strength of 50–500 mM. Divalent cations can reduce non-specific adsorption caused by ionic and hydrophobic interactions; for example, 0–50 mM MgCl sub>2 can be added to the loading buffer. Mg2+ can also inhibit charge repulsion between the resin and phosphate-containing substances such as nucleic acids, and adding Mg2+ can enhance the binding effect.When the formed boronate ester exists as an uncharged planar triangular structure, the boron atom has an empty orbital and can act as an electron acceptor for charge-transfer interactions. Unprotonated amino groups are good electron donors; when the amino group provides a lone pair of electrons to the boron atom, the boron atom forms a tetrahedral shape, promoting the formation of boronate ester. However, it should be noted that when a hydroxyl group exists near the amino group, the resin cannot form a boronate ester structure with 1,2-cis-diol. This is why ethanolamine and Tris are not suitable as loading buffers.Figure 1. Affinity principle of boronate affinity chromatography mediumInstructions for Use1. Column PackingThe column packing method for the resin connected to the chromatography system is described below.(1)Equilibrate all materials to the same temperature as the chromatography operation; liquids are preferably degassed.(2)Resin dosage calculation: Quantify the resin volume by sedimentation. Required settled resin volume = Column volume × Compression factor. The settled volume is the stable volume read after complete natural sedimentation of the resin in 20% ethanol storage solution. The compression factor of boronate affinity chromatography medium is ~1.10. To achieve the compression factor, compression can be performed by lowering the column top or at a high flow rate.(3)Resin washing: Mix the resin suspension thoroughly, measure the corresponding volume, remove the liquid, and wash with approximately 3 resin volumes of purified water, repeated 3 times to remove the storage solution.(4)Packing slurry preparation: Transfer the resin to a suitable container, add an appropriate amount of packing buffer to prepare a 50–75 v% packing slurry (the original package contains 50% resin by volume), and mix well before use.(5)Pre-packing preparation: Add packing buffer to the bottom of the cleaned chromatography column to remove air from the lower frit and the column bottom, leave a small amount of distilled water in the column, tighten the lower plug, and adjust the column to be vertical to the ground.(6)Packing: Pour the well-mixed resin suspension slowly into the chromatography column at one time (use a packing device if necessary). To avoid introducing air bubbles, let it flow naturally along the inner wall of the chromatography column. After adding all the resin, fill the packing device with packing buffer, tighten the top cover of the packing device, and connect the column to the chromatography system.(7)Compaction: Let the resin settle naturally (or at a low flow rate of 50–150 cm/h). After complete sedimentation (clear interface between resin and liquid), remove the packing device, install the upper column top, and lower the top to the interface; continue compaction at a high flow rate (see the table below for recommended flow rate; note that column pressure does not exceed 0.3 MPa) until the interface is clear and stable, and mark the column height when the interface is stable. Stop the pump, open the valve/plug on the column top, close the valve/plug at the column bottom, lower the top to the position below the marked position corresponding to the compression factor, tighten the top, and column packing is completed. After packing, equilibrate the resin in the column at a high flow rate.Packing ConditionsBoronate Affinity Chromatography ResinCompression factor~1.10Packing flow rate600–1500 cm/h, depending on column size, subject to compression factor and column efficiency test2. Column Efficiency Determination and EvaluationColumn efficiency can be measured and evaluated to confirm the packing quality after packing and before use. Column efficiency is usually evaluated by Height Equivalent to a Theoretical Plate (HETP) and Asymmetry factor (As).Column efficiency can be determined using acetone or NaCl as samples. Prepare sample solutions and mobile phases according to the table below.Item1.0% Acetone0.8–1.0 M NaClSample volume1.0% column volume1.0% column volumeMobile phasePure water0.4 M NaClFlow rate30 cm/h30 cm/hDetectorUV-280 nmConductivityCalculate HETP, Number of theoretical plates (N) and As based on UV or conductivity curves using the following formulas:HETP = L / NN = 5.54 × (Vr / Wh)2As = a / bWhere:L = column height; Vr = retention volume; Wh = peak width at half height; a = first half-peak width at 10% peak height; b = second half-peak width at 10% peak height.Generally, HETP should be less than three times the mean particle size of the resin (i.e., HETP/D50 < 3, D50 = mean particle size of the resin), and As should be between 0.8 and 1.5.3. Equilibration and Sample LoadingBefore sample loading, equilibrate the column with loading buffer (equilibration buffer) at the operating flow rate, and observe changes in the detector until parameters such as conductivity and pH remain constant. The sample loading volume is selected according to the sample properties and the amount of chromatography medium, which can be determined in preliminary experiments (e.g., static adsorption experiment). Generally, the sample needs to be diluted with loading buffer, e.g., 1:1 dilution.The pH of the loading buffer is usually 8.5–9.0. For example, HEPES buffer (20 mM HEPES, 20 mM MgCl2, pH 8.5) can be used to separate glycoproteins from non-glycoproteins, and ammonium acetate buffer (0.25 M ammonium acetate, pH 8.8) can be used to separate ribonucleosides from deoxyribonucleosides. Common loading buffer systems for this resin include phosphate, ammonium acetate, HEPES, etc. In general, amino-containing buffers such as Tris-HCl should be avoided.4. ElutionWash the loaded column with 2–3 column volumes of equilibration buffer, and observe changes in the detector until parameters such as conductivity and pH remain constant. At this time, unbound components are washed out.Elution of affinity chromatography columns can be performed by isocratic elution, gradient elution or step elution. During elution, the pH can be reduced to 4–6 (e.g., 0.1 M formic acid, 25 mM hydrochloric acid); sugars can also be selected for competitive elution, such as 10–200 mM sorbitol; 0–20 mM EDTA can also be used; Tris can be used as a high-efficiency eluent.5. Regeneration and Cleaning-in-Place (CIP)The resin can usually be regenerated with 5 CV of eluent. If inactivated proteins or lipids cannot be washed out during regeneration, they can be removed by Cleaning-in-Place (CIP). The following options can be used for CIP: 0.5 mol/L NaOH, 70% ethanol, 30% isopropanol, etc. CIP can effectively remove impurities on the medium, and the effect is better in reverse flow. Refer to the table below for specific CIP solution selection.CIP ProcessPurpose0.5 mol/L NaOH (minimize cleaning time); water, 5 CVRemove protein precipitates or other impurities such as pyrogens30% isopropanol or 70% ethanol, 4–10 CV, <50 cm/h; water, 5 CVRemove tightly bound lipoproteins, liposomes, etc.6. Resin StorageThe initial storage solution of the resin is 20% ethanol, and the resin can continue to be stored in 20% ethanol after use. It can also be stored in buffers similar to elution and regeneration, such as 0.2 M sodium acetate and 0.1 M formic acid. 0.02% sodium azide can be added to the buffer for antisepsis. The resin is more stable in a weakly acidic environment. The storage temperature is preferably 2–8 °C; do not freeze.

Grade: BioReagent

Storage Temperature: Store at 2-8°C;Do not freeze

Shipped in: Wet ice;Do not freeze

UPC:

51441701

Weight:

9.87 Ounces

HazmatClass:

No

Quantity:

1

Unit of Measurement:

EA

WeightUOM:

LB

MPN:

B1523011-100ml

Country of Origin:

China

Product Size:

100ml

Harmonized Tariff Code:

2931.90.90