CNBr-activated Agrose 4FF (C008B-269586)

CNBr-activated Agrose 4FF is a pre-activated agarose medium with 4% highly cross-linked agarose as the matrix, featuring excellent physical and chemical stability. The medium enables simple, direct, rapid and mild coupling, and can directly conjugate proteins and other biological macromolecules containing primary amino groups without a coupling spacer arm. It also allows multi-point coupling, making ligands not easy to detach. Meanwhile, it has strong hydrophilicity, low non-specific adsorption, wide universality and application scope, fast flow rate, good repeatability and easy process scale-up. It is mainly used for the separation and purification of biological molecules such as proteins.Aladdin  CNBr-activated Agrose 4FF is stored in acetone, with a volume ratio of gel to protective solution of 7:3. The product specification is based on the actual gel volume.Parameter SpecificationsItemSpecificationMatrix4% Highly cross-linked agaroseCoupling functional group-NH₂Activated groupCNBrAverage particle size90 μmCoupling conditionspH 8–10, room temperature (20–25℃) for 2–3 h or 4℃ overnightCoupling capacity13–26 mg α-chymotrypsinogen/mL mediumRecommended flow rate<250 cm/hRecommended pressure<0.15 MPa (1.5 bar)pH stability ①3–11 (long-term); 2–11 (short-term )Chemical stabilityStable in common aqueous buffers after ligand coupling; denaturants such as 8 M urea or 6 M guanidine hydrochloride can be used ②Storage conditionsAcetone; below -15℃Shelf life1 yearNotes:① Long-term refers to the pH range where the medium remains stable for a long time without adverse effects on its subsequent performance. Short-term refers to the pH range for medium regeneration, cleaning-in-place and disinfection based on experience.② The stability after ligand coupling is premised on the ligand’s tolerance to changes in pH or chemical environment.Instructions for Use1. Coupling of Functional Ligands1.1 Buffer VolumeSolutionCompositionVolume RatioSolution A1 mM HCl, 0.5 M NaCl (pre-cooled, pH 2–3)≥20× gel volumeSolution B0.2 M NaHCO₃, 0.5 M NaCl, pH 8.3≥10× gel volumeSolution C0.1 M Tris-HCl, pH 8.0≥5× gel volumeSolution D0.05 M Tris-HCl, 0.5 M NaCl, pH 8–9≥15× gel volumeSolution E0.1 M Acetic acid-sodium acetate, 0.5 M NaCl, pH 3–4≥15× gel volumeSolution F0.02 M PB, 0.15 M NaCl, pH 7.0–7.6≥10× gel volume1.2 Dissolve dry powder protein ligand with Solution B; replace the buffer of solution-state protein ligand with Solution B.1.3 Place an appropriate amount of affinity chromatography medium stored in acetone in a glass filter funnel, and wash with 15× gel volume of pre-cooled Solution A.1.4 After washing, add the medium to the ligand solution, mix well, and react at room temperature for 2–4 h or at 4℃ overnight.1.5 After coupling, wash with 5× gel volume of Solution B to remove excess uncoupled ligand.1.6 Wash with 3× gel volume of Solution C, then continue to block remaining active sites of the medium with Solution C at room temperature for 2–4 h.1.7 Wash alternately with Solution D and Solution E for 3–6 cycles, 3× gel volume each time.1.8 Finally, wash with 10× gel volume of Solution F to complete ligand coupling.Coupling NotesCoupling buffer: Bicarbonate or borate buffer is commonly used. Do not use Tris or other amino-containing buffers as coupling buffer; pH 8–10 is recommended for high coupling efficiency. Low pH affects coupling activity, but pH 6 may be used to reduce steric hindrance or preserve ligand bioactivity.Add 0.5 M NaCl to reduce protein-protein adsorption.Temperature & time: 2–3 h at room temperature (25℃); overnight (10–16 h) at 4℃.Ligand concentration: 5–10 mg protein per mL medium is recommended. Excess ligand causes high ligand density, steric hindrance, reduced binding capacity, difficult elution and increased non-specific adsorption.2.Column Packing2.1 Packing Buffer PreparationPurified water, ultrasonic degassing for 15 min.2.2 Chromatography Medium PreparationCalculate required medium volume (compression factor ~1.15) and weigh. Replace buffer to packing buffer via vacuum filtration. Prepare ~75% gel suspension with packing buffer.2.3 Chromatography Column PreparationInspect column for integrity and cleanliness. Install lower end cap, tighten O-ring, fix column vertically and verify with level gauge. Remove air bubbles from bottom sieve with packing buffer via syringe, then cap. Add ~2 cm height of packing buffer to column.2.4 Column Packing (Example: 16 mm diameter, 15 cm bed height)Mix gel suspension, pour slowly into column with glass rod; fill with packing buffer if needed. Connect regulator to system, purge air bubbles at set flow rate then pause. Insert regulator at 45° and seal. Open bottom cap, set flow rate to 60 cm/h until bed interface stabilizes, then increase to 400 cm/h for 45 min, mark interface. Pause system, cap bottom, disconnect top, loosen regulator seal, lower upper end cap to 2 mm below gel surface, retighten. Connect column to system for column efficiency test.3.Column Efficiency Determination and EvaluationColumn efficiency should be tested immediately after packing. Column performance is usually evaluated by theoretical plates per meter (N/m) and peak asymmetry factor (As). Higher column efficiency indicates stronger separation ability. For accurate results, the sample volume is 1.0% of column volume, and linear flow rate is controlled at 15–30 cm/h. Load the sample close to the column inlet to minimize dilution.ItemAcetone MethodNaCl MethodSample1.0% (v/v) aqueous acetone0.8 M NaCl aqueous solutionSample volume1.0% column volume1.0% column volumeMobile phasePure water0.4 M NaCl aqueous solutionFlow rate30 cm/h30 cm/hDetectorUV-280 nmConductivityColumn Efficiency CalculationCalculate N/m and As from the UV (or conductivity) curve using the following formulas:N/m = N/LN = 5.54(VR/Wh)2 As = b/aNotes: 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.As should be between 0.8 and 1.8. N/m > 3000.4. Separation and Purification4.1 Column EquilibrationFlush column with binding buffer for ≥3–5 CV until effluent pH and conductivity match binding buffer; zero UV detector.4.2 Sample LoadingLoad sample filtered through 0.2/0.45 μm membrane. Loading volume depends on medium capacity, target molecule concentration and conditions.4.3 Column WashingWash with binding buffer for 3–5 CV until UV 280 returns to baseline (or use optimized washing conditions).4.4 ElutionElute with elution buffer; collect fractions when target UV peak appears.4.5 Cleaning and StorageRegeneration: Alternate high-pH (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5) and low-pH (0.1 M sodium acetate-acetic acid, 0.5 M NaCl, pH 4.5) buffers for 3–5 cycles (2–3 CV each). Equilibrate with binding buffer (3–5 CV), flush with pure water to zero conductivity, then flush with 20% ethanol (2–3 CV). Store at 2–8℃.Conditions vary by ligand; all buffers must be 0.22 μm filtered. Sample buffer should match equilibration buffer.5. Cleaning-In-Place (CIP)CIP removes strongly bound, precipitated or denatured contaminants that impair performance, increase backpressure and reduce flow rate. Recommended every 1–5 cycles.Precipitated/denatured contaminants: 0.1 M NaOH (4 CV, 1–2 h contact) → binding buffer (≥5 CV); or 6 M guanidine hydrochloride (2 CV) → immediate binding buffer wash (≥5 CV).Hydrophobic impurities: 0.1–0.5% non-ionic detergent (2 CV) → binding buffer (≥5 CV); or 70% ethanol (3–4 CV) → immediate binding buffer wash (≥5 CV).6. SterilizationMinimize microbial contamination with 70% ethanol (if ligand-tolerant) for 12 h, then flush with sterile binding buffer (≥5 CV).7. StorageUnused medium: Store below -15℃, protected from light, dry, ventilated and sealed; short-term storage at 4℃ (light-proof).Coupled medium/column: Store sealed in 20% ethanol or 2% benzyl alcohol. Replace storage solution every 3 months to prevent microbial growth.8. Linear Scale-UpLab-scale purification can be linearly scaled to pilot or production scale with these guidelines:Keep residence time constant for stable dynamic binding capacity.Select column volume based on binding demand; adjust bed height if needed.Determine column diameter by flow rate; recommended bed height: 3–15 cm.Maintain uniform sample concentration and consistent elution conditions.

Specifications & Purity: CNBr-activated Agrose 4FF is a pre-activated agarose medium with 4% highly cross-linked agarose as the matrix, featuring excellent physical and chemical stability. The medium enables simple, direct, rapid and mild coupling, and can directly conjugate proteins and other biological macromolecules containing primary amino groups without a coupling spacer arm. It also allows multi-point coupling, making ligands not easy to detach. Meanwhile, it has strong hydrophilicity, low non-specific adsorption, wide universality and application scope, fast flow rate, good repeatability and easy process scale-up. It is mainly used for the separation and purification of biological molecules such as proteins.Aladdin  CNBr-activated Agrose 4FF is stored in acetone, with a volume ratio of gel to protective solution of 7:3. The product specification is based on the actual gel volume.Parameter SpecificationsItemSpecificationMatrix4% Highly cross-linked agaroseCoupling functional group-NH₂Activated groupCNBrAverage particle size90 μmCoupling conditionspH 8–10, room temperature (20–25℃) for 2–3 h or 4℃ overnightCoupling capacity13–26 mg α-chymotrypsinogen/mL mediumRecommended flow rate<250 cm/hRecommended pressure<0.15 MPa (1.5 bar)pH stability ①3–11 (long-term); 2–11 (short-term )Chemical stabilityStable in common aqueous buffers after ligand coupling; denaturants such as 8 M urea or 6 M guanidine hydrochloride can be used ②Storage conditionsAcetone; below -15℃Shelf life1 yearNotes:① Long-term refers to the pH range where the medium remains stable for a long time without adverse effects on its subsequent performance. Short-term refers to the pH range for medium regeneration, cleaning-in-place and disinfection based on experience.② The stability after ligand coupling is premised on the ligand’s tolerance to changes in pH or chemical environment.Instructions for Use1. Coupling of Functional Ligands1.1 Buffer VolumeSolutionCompositionVolume RatioSolution A1 mM HCl, 0.5 M NaCl (pre-cooled, pH 2–3)≥20× gel volumeSolution B0.2 M NaHCO₃, 0.5 M NaCl, pH 8.3≥10× gel volumeSolution C0.1 M Tris-HCl, pH 8.0≥5× gel volumeSolution D0.05 M Tris-HCl, 0.5 M NaCl, pH 8–9≥15× gel volumeSolution E0.1 M Acetic acid-sodium acetate, 0.5 M NaCl, pH 3–4≥15× gel volumeSolution F0.02 M PB, 0.15 M NaCl, pH 7.0–7.6≥10× gel volume1.2 Dissolve dry powder protein ligand with Solution B; replace the buffer of solution-state protein ligand with Solution B.1.3 Place an appropriate amount of affinity chromatography medium stored in acetone in a glass filter funnel, and wash with 15× gel volume of pre-cooled Solution A.1.4 After washing, add the medium to the ligand solution, mix well, and react at room temperature for 2–4 h or at 4℃ overnight.1.5 After coupling, wash with 5× gel volume of Solution B to remove excess uncoupled ligand.1.6 Wash with 3× gel volume of Solution C, then continue to block remaining active sites of the medium with Solution C at room temperature for 2–4 h.1.7 Wash alternately with Solution D and Solution E for 3–6 cycles, 3× gel volume each time.1.8 Finally, wash with 10× gel volume of Solution F to complete ligand coupling.Coupling NotesCoupling buffer: Bicarbonate or borate buffer is commonly used. Do not use Tris or other amino-containing buffers as coupling buffer; pH 8–10 is recommended for high coupling efficiency. Low pH affects coupling activity, but pH 6 may be used to reduce steric hindrance or preserve ligand bioactivity.Add 0.5 M NaCl to reduce protein-protein adsorption.Temperature & time: 2–3 h at room temperature (25℃); overnight (10–16 h) at 4℃.Ligand concentration: 5–10 mg protein per mL medium is recommended. Excess ligand causes high ligand density, steric hindrance, reduced binding capacity, difficult elution and increased non-specific adsorption.2.Column Packing2.1 Packing Buffer PreparationPurified water, ultrasonic degassing for 15 min.2.2 Chromatography Medium PreparationCalculate required medium volume (compression factor ~1.15) and weigh. Replace buffer to packing buffer via vacuum filtration. Prepare ~75% gel suspension with packing buffer.2.3 Chromatography Column PreparationInspect column for integrity and cleanliness. Install lower end cap, tighten O-ring, fix column vertically and verify with level gauge. Remove air bubbles from bottom sieve with packing buffer via syringe, then cap. Add ~2 cm height of packing buffer to column.2.4 Column Packing (Example: 16 mm diameter, 15 cm bed height)Mix gel suspension, pour slowly into column with glass rod; fill with packing buffer if needed. Connect regulator to system, purge air bubbles at set flow rate then pause. Insert regulator at 45° and seal. Open bottom cap, set flow rate to 60 cm/h until bed interface stabilizes, then increase to 400 cm/h for 45 min, mark interface. Pause system, cap bottom, disconnect top, loosen regulator seal, lower upper end cap to 2 mm below gel surface, retighten. Connect column to system for column efficiency test.3.Column Efficiency Determination and EvaluationColumn efficiency should be tested immediately after packing. Column performance is usually evaluated by theoretical plates per meter (N/m) and peak asymmetry factor (As). Higher column efficiency indicates stronger separation ability. For accurate results, the sample volume is 1.0% of column volume, and linear flow rate is controlled at 15–30 cm/h. Load the sample close to the column inlet to minimize dilution.ItemAcetone MethodNaCl MethodSample1.0% (v/v) aqueous acetone0.8 M NaCl aqueous solutionSample volume1.0% column volume1.0% column volumeMobile phasePure water0.4 M NaCl aqueous solutionFlow rate30 cm/h30 cm/hDetectorUV-280 nmConductivityColumn Efficiency CalculationCalculate N/m and As from the UV (or conductivity) curve using the following formulas:N/m = N/LN = 5.54(VR/Wh)2 As = b/aNotes: 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.As should be between 0.8 and 1.8. N/m > 3000.4. Separation and Purification4.1 Column EquilibrationFlush column with binding buffer for ≥3–5 CV until effluent pH and conductivity match binding buffer; zero UV detector.4.2 Sample LoadingLoad sample filtered through 0.2/0.45 μm membrane. Loading volume depends on medium capacity, target molecule concentration and conditions.4.3 Column WashingWash with binding buffer for 3–5 CV until UV 280 returns to baseline (or use optimized washing conditions).4.4 ElutionElute with elution buffer; collect fractions when target UV peak appears.4.5 Cleaning and StorageRegeneration: Alternate high-pH (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5) and low-pH (0.1 M sodium acetate-acetic acid, 0.5 M NaCl, pH 4.5) buffers for 3–5 cycles (2–3 CV each). Equilibrate with binding buffer (3–5 CV), flush with pure water to zero conductivity, then flush with 20% ethanol (2–3 CV). Store at 2–8℃.Conditions vary by ligand; all buffers must be 0.22 μm filtered. Sample buffer should match equilibration buffer.5. Cleaning-In-Place (CIP)CIP removes strongly bound, precipitated or denatured contaminants that impair performance, increase backpressure and reduce flow rate. Recommended every 1–5 cycles.Precipitated/denatured contaminants: 0.1 M NaOH (4 CV, 1–2 h contact) → binding buffer (≥5 CV); or 6 M guanidine hydrochloride (2 CV) → immediate binding buffer wash (≥5 CV).Hydrophobic impurities: 0.1–0.5% non-ionic detergent (2 CV) → binding buffer (≥5 CV); or 70% ethanol (3–4 CV) → immediate binding buffer wash (≥5 CV).6. SterilizationMinimize microbial contamination with 70% ethanol (if ligand-tolerant) for 12 h, then flush with sterile binding buffer (≥5 CV).7. StorageUnused medium: Store below -15℃, protected from light, dry, ventilated and sealed; short-term storage at 4℃ (light-proof).Coupled medium/column: Store sealed in 20% ethanol or 2% benzyl alcohol. Replace storage solution every 3 months to prevent microbial growth.8. Linear Scale-UpLab-scale purification can be linearly scaled to pilot or production scale with these guidelines:Keep residence time constant for stable dynamic binding capacity.Select column volume based on binding demand; adjust bed height if needed.Determine column diameter by flow rate; recommended bed height: 3–15 cm.Maintain uniform sample concentration and consistent elution conditions.

Grade: BioReagent

Storage Temperature: Store at -20°C

Shipped in: Ice chest + Ice pads

UPC:

51284138

Weight:

2.64 Ounces

HazmatClass:

No

Quantity:

1

Unit of Measurement:

EA

WeightUOM:

LB

MPN:

C1523222-25ml

Country of Origin:

China

Product Size:

25ml

Harmonized Tariff Code:

2931.90.90