Affinity Chromatography (AC) is a chromatographic technique that separates molecules based on specific biological affinities. When a sample passes through the stationary phase, the target biomolecule binds specifically to the immobilized ligand, while impurities flow through with the eluent, enabling the separation and purification of the biomolecule. Affinity chromatography is one of the most commonly used techniques for purifying proteins, enzymes, and other macromolecules, offering a simple and rapid separation process. It is also frequently used in the study of macromolecular structure and function.
This product is a thiophilic affinity chromatography medium prepared by coupling 2-mercaptopyridine to a highly rigid agarose matrix. Thiophilic affinity chromatography separates and purifies biomolecules based on electron donor-acceptor interactions, which are enhanced under high-salt conditions and weakened under low-salt conditions. This medium is primarily used for the purification of supercoiled plasmid DNA. During purification, supercoiled plasmid DNA adsorbs onto the medium, while open-circular plasmid DNA does not bind, achieving effective separation. The resulting high-quality supercoiled plasmid DNA is commonly used in gene therapy or as a DNA vaccine.
Aladdin Plasmid Agarose Resin is stored in 20% ethanol, with a settled gel to storage solution ratio of 7:3. The product specification refers to the actual volume of the settled gel.
Parameter
| Specification
|
Matrix
| Highly Rigid Agarose
|
| Ligand | 2-Mercaptopyridine
|
| Average Particle Size | 40 μm
|
Binding Capacity
| ~3 mg supercoiled plasmid DNA / mL medium
|
Recommended Flow Rate
| ＜300 cm/h
|
Recommended Pressure
| ＜0.5 MPa (5 bar)
|
pH Stability¹
| 3-11 (long term); 2-13 (short term)
|
Chemical Stability
| Stable in common aqueous buffers (e.g., 70% ethanol, 0.1 M NaOH, 1 M acetic acid)
|
| Storage | In 20% Ethanol, 4-30°C |
Note1:
Long term: pH range where the medium remains stable for extended periods without adversely affecting its subsequent performance.
Short term: pH range empirically recommended for regeneration, cleaning-in-place (CIP), and sanitization.
Instructions for Use
1. Column Packing
1.1 Packing Buffer Preparation
Prepare purified water. Degas by sonication for 15 min.
1.2 Chromatography Medium Preparation
Calculate the required amount of medium (compression factor ~1.15) and weigh it out. Transfer it to the packing buffer using a vacuum filtration flask. Add an appropriate amount of packing buffer to the exchanged medium to create a ~50% slurry.
1.3 Chromatography Column Preparation
Inspect the column to ensure all parts are intact and clean. Attach the bottom adaptor, tighten the O-ring, and secure the column vertically on a stand. Check and adjust for vertical alignment using a level. Use a syringe to draw packing buffer, connect it to the bottom outlet, and slowly push the liquid to remove air bubbles from the bottom screen. Remove the syringe and attach the end cap. Add packing buffer to the column to a height of ~2 cm.
1.4 Packing (Example for a column with diameter 16 mm, bed height 10 cm)
Mix the slurry thoroughly. Slowly pour it into the column using a glass rod as a guide. If there is space left at the top, fill it with packing buffer. Connect the adaptor to the chromatography system. Start the pump to purge air bubbles from the tubing and top screen at a certain flow rate, then pause. Insert the adaptor into the column at a 45° angle, secure it, and tighten the seal, avoiding air bubbles.
Unscrew the bottom end cap and place the outlet tubing into a waste container. Set the flow rate to 60 cm/h until the bed interface stabilizes. Then set the flow rate to 1200 cm/h and maintain for 45 min. Mark the interface with a pen and pause the system. Attach the bottom end cap. Disconnect the top of the column from the pump. Slightly loosen the adaptor seal, lower the adaptor to 1-3 mm below the bed surface, and tighten the seal. Connect the top and bottom tubing of the column to the chromatography system for column efficiency testing.
2. Column Efficiency Test
Column efficiency should be tested immediately after packing. Terms such as Number of Theoretical Plates per meter (N/m) and Peak Asymmetry Factor (As) are commonly used to evaluate column performance. Higher column efficiency indicates better separation capability. For accurate assessment, the sample volume should be 1.0% of the column volume (CV), and the linear flow rate should be controlled between 15-30 cm/h. To prevent sample dilution, inject the sample as close to the column inlet as possible.
Method
| Acetone Test
| NaCl Test
|
Equilibration Buffer
| Purified Water
| 0.4 M NaCl
|
Sample
| 1% Acetone in H₂O, 1.0% CV
| 0.8 M NaCl, 1.0% CV
|
Flow Rate
| 30 cm/h
| 30 cm/h
|
Detection Signal
| UV 280 nm
| Conductivity
|
| Note: CV (Column Volume) | |
Column Efficiency Calculation
Calculate N/m and As from the UV curve (or conductivity curve) using the following formulas:
N/m = N / L
N = 5.54 (Vʀ / Wₕ)²
As = b / a
Where:
Vʀ: Retention Volume
Wₕ: Peak Width at Half Height
L: Column Height (in meters)
N: Number of Theoretical Plates
Vʀ and Wₕ must have the same units.
a: Width of the first half of the peak at 10% peak height
b: Width of the second half of the peak at 10% peak height
The peak should be symmetrical, with As as close to 1 as possible (values between 0.8 and 1.8 are generally acceptable). N/m should be > 9,000.
3. Separation and Purification
3.1 Column Equilibration
Connect the column to the purification system. Equilibrate with Binding Buffer for more than 2-3 CV until the pH and conductivity of the column effluent match those of the Binding Buffer. Zero the UV detector after equilibration.
*Note: Binding Buffer: 100 mM Tris-HCl, 2.1 M (NH₄)₂SO₄, 10 mM EDTA, pH 7.5, filtered through 0.2 μm.*
3.2 Sample Loading
Load the sample containing supercoiled plasmid DNA (filtered through a 0.45 μm membrane) onto the column. The loading volume depends on the medium's binding capacity, the concentration of supercoiled plasmid DNA in the sample, and other chromatographic conditions. Samples treated for RNA removal can be loaded directly. Other samples may require salt and acid (or base) adjustment to match the conductivity and pH of the Binding Buffer before filtration (0.45 μm) and loading.
*Note: Recommended sample loading flow rate < 150 cm/h. Slower flow rates are needed for taller columns.*
3.3 Column Washing
Wash the column with 3-5 CV of Binding Buffer to remove impurities that do not bind under these conditions (until the UV 280 signal returns to baseline). Alternatively, use washing conditions determined from prior experiments.
3.4 Elution
Elute the column using Elution Buffer. Start collecting fractions when the target UV peak begins to rise.
*Note: Elution Buffer: 100 mM Tris-HCl, 1.7 M (NH₄)₂SO₄, 10 mM EDTA, 0.3 M NaCl, pH 7.5, filtered through 0.2 μm.*
3.5 Cleaning and Storage
Wash the column with 3 CV of purified water, followed by 3-5 CV of 0.5 M NaOH. Then rinse with purified water until neutral pH is reached. Finally, rinse with 2-3 CV of 20% ethanol. Store the column at 4-30°C.
4. Cleaning In Place (CIP)
CIP removes strongly bound, precipitated, or denatured substances from the chromatography medium. Residual impurities can affect column performance. Severe buildup can clog the column, increasing backpressure and reducing flow rate. Regular CIP prevents contaminant accumulation and helps maintain the medium's binding capacity and flow properties. Common CIP methods:
4.1 Removing Denatured Proteins: Wash the column with 3-5 CV of 0.5 M NaOH, then immediately flush with at least 5 CV of Binding Buffer.
4.2 Removing Strongly Hydrophobic Proteins or Lipids: Wash the column with 3-5 CV of 20 mM PB, 30% Isopropanol, pH 7.5, then quickly flush with at least 5 CV of purified water.
5. Sanitization
Sanitization minimizes microbial contamination in the column. Due to its high alkali resistance, NaOH solution can be used as a sanitizing agent for this medium. NaOH effectively removes viruses, bacteria, yeast, and endotoxins at very low cost. Sanitization procedure:
5.1 Flush the column with 3-5 CV of Binding Buffer.
5.2 Flush the column with 2-5 CV of 0.5 M NaOH solution.
5.3 Soak the column in 0.1 M NaOH for 30 minutes (or 0.5 M NaOH for 15 minutes).
5.4 Flush the column with 5-10 CV of Binding Buffer (pH 7-8) to complete the sanitization process.
*Precautions: For heavily contaminated columns, use 0.5 M NaOH containing 30-40% propanol for cleaning. High NaOH concentrations or prolonged exposure can reduce medium binding capacity; pay attention to concentration and time during sanitization.*
6. Storage
Unused medium should be stored in a dry, ventilated, clean environment at 4-30°C, ensuring the container is tightly sealed. Do not freeze.
Packed columns should be stored submerged in 20% ethanol or 2% benzyl alcohol, sealed, at 4-30°C to prevent microbial growth. Before use, perform a blank run and CIP.
To prevent microbial growth due to ethanol evaporation, replace the storage solution with fresh solution every 3 months.
Note: Before using stored medium, perform CIP and equilibrate with at least 5 CV of Binding Buffer.
7. Linear Scale-Up
Purification processes optimized at laboratory scale can be linearly scaled up to pilot or production scale. Consider the following during scale-up:
7.1 Maintain the same residence time to ensure consistent dynamic binding capacity.
7.2 Select the column volume based on the required binding capacity. Note if changes in bed height affect the purification steps.
7.3 Determine the column diameter based on flow rate requirements. Determine the bed height based on the known residence time; 10-25 cm is generally recommended.
7.4 Ensure sample concentration homogeneity and consistent elution conditions.
Troubleshooting
