Dextrin affinity chromatography resin is an affinity chromatography separation medium formed by covalently coupling dextrin ligands to agarose beads. The dextrin ligand specifically binds to maltose-binding protein (MBP), enabling the purification of MBP-tagged fusion proteins. This product allows target protein purification under near-physiological conditions, maximally preserving the biological activity of the target protein. The MBP tag is relatively large, with a molecular weight of approximately 42 kDa. Its main functions include promoting the correct folding of the target protein, enhancing protein expression levels, and improving the solubility of the target protein. Meanwhile, MBP-tagged fusion proteins are compatible with specific affinity resins, and the MBP tag is widely used in Escherichia coli expression systems. After affinity chromatography, the tag is usually removed by site-specific proteases. The binding capacity of this resin is 3-5 mg of MBP fusion protein per milliliter of settled gel. The particle size of Dextrin Resin ranges from 45 to 165 μm, and the maximum withstand pressure is 0.3 MPa. Dextrin affinity chromatography resin is supplied in 20% ethanol, with a 1:1 volume ratio of gel to storage solution. For a total volume of 10 mL, 5 mL is settled gel and 5 mL is storage solution. The gel should be fully resuspended before pipetting during use.
Product Features
1. Fast and Efficient: This kit utilizes MBP-tagged protein purification resin with high binding efficiency. It shortens the operation time and effectively prevents degradation or denaturation of the target protein during prolonged handling, thereby fully preserving the activity of the target protein.
2. Convenient: All necessary reagents and empty affinity chromatography columns for MBP-tagged protein purification are provided, greatly facilitating the purification process of MBP-tagged proteins.
3. High Specificity: The dextrin ligand specifically binds to maltose-binding protein (MBP), enabling one-step purification of the target protein.
4. No Impact on Protein Activity: Mild elution is performed using maltose, without the use of detergents or denaturants that would affect protein activity.
Instruction for use
1 Sample Preparation
1.1 Soluble Protein Expressed in Bacteria or Yeast
1.1.1 Pick a single colony into medium containing the appropriate antibiotic. Induce expression with inducer at the recommended concentration and duration according to the vector instructions.
1.1.2 After expression, transfer the culture to a centrifuge bottle and centrifuge at 7000 rpm (7500 ×g) for 15 min to harvest cells. Resuspend the cell pellet in Native Lysis Buffer at a ratio of 1:10 (w/v). Optionally add PMSF to a final concentration of 1 mM or other protease inhibitors if the protein is prone to degradation, provided they do not interfere with binding to the resin. Lysozyme may be added as needed (not required if the host strain contains pLysS or pLysE).
1.1.3 Fully resuspend the cell pellet. If the cell suspension is concentrated, add 10 μg/mL RNase A and 5 μg/mL DNase I. Mix well, keep on ice, and lyse by sonication in an ice-water bath until the lysate becomes clear.
1.1.4 Transfer the lysate to a centrifuge tube and centrifuge at 10000 rpm (15000 ×g) at 4 °C for 20-30 min. Collect the supernatant, keep on ice, and proceed to purification.
1.2 Soluble Protein Expressed in Yeast, Insect, or Mammalian Cells
Transfer the cell culture to a centrifuge bottle and centrifuge at 5000 rpm (3800 ×g) for 10 min. Collect the supernatant, which may be loaded directly. For large volumes, concentrate by ammonium sulfate precipitation and dialyze against equilibration buffer before loading.
2 Buffer Preparation
Preparation of Elution Buffer Containing Maltose Monohydrate: The elution buffer is supplied without maltose. Add 360 mg of maltose monohydrate to 100 mL of elution buffer to prepare elution buffer containing 10 mM maltose. After preparation, filter the solution through a 0.22 μm or 0.45 μm filter and store at 2-8°C. Prepare according to experimental consumption; fresh preparation is recommended.
3 Sample Purification
Before loading, samples are recommended to be centrifuged or filtered through a 0.22 μm or 0.45 μm filter to reduce impurities, improve purification efficiency, and prevent column clogging. Buffers are also recommended to be filtered through a 0.22 μm or 0.45 μm filter before use.
3.1 Gravity Column Purification
3.1.1 Column Packing
3.1.1.1 Take an empty gravity column, insert the bottom frit, rinse with purified water, and close the bottom outlet.
3.1.1.2 Mix the Dextrin Resin thoroughly and pipette the slurry into the gravity column. The settled resin volume is approximately half the slurry volume (e.g., 1 mL slurry yields ~0.5 mL resin, with a maximum capacity of ~10 mg). Open the outlet to drain the storage solution.
3.1.1.3 Wash the resin with purified water. Drain completely and close the outlet.
3.1.1.4 Insert the top frit, ensuring no air gap between frit and resin and that the frit is level.
3.1.1.5 The packed column may be equilibrated immediately with lysis buffer/wash buffer. If not used immediately, add storage solution and store at 4 °C.
3.1.2 Equilibration
Equilibrate the column with 2-5 CV Lysis Buffer/Wash Buffer (CV = settled resin volume; this definition applies throughout the protocol). Repeat 2-3 times.
3.1.3 Sample Loading
Apply the sample to the equilibrated column. Allow the sample to flow slowly to ensure sufficient binding. Collect the flow-through; reloading may increase binding efficiency. Keep samples on ice if the target protein is unstable.
3.1.4 Washing
Wash with 5-10 CV Wash Buffer to remove non‑specifically bound proteins. Collect the wash fraction.During washing and subsequent elution, protein concentration in each wash and elution fraction can be rapidly monitored using the Bradford method to adjust the number of washing and elution steps as needed.
3.1.5 Elution
Elute the target protein with 5-10 CV Elution Buffer Containing Maltose Monohydrate. Collect fractions every 1-5 CV and analyze individually to ensure complete elution and obtain highly pure, concentrated protein. After elution, wash with 3 CV equilibration buffer, 5 CV purified water, and 2 CV storage solution. Store the resin at 2-8 °C.
3.2 Batch Purification
3.2.1 Resin Preparation
Transfer an appropriate volume of Dextrin Resin into a centrifuge tube. Centrifuge at 1000 rpm for 1 min and carefully remove the supernatant (use low speed to avoid damaging the resin). Alternatively, drain the storage solution in a gravity column.
3.2.2 Resin Equilibration
Add 5 CV Lysis Buffer/Wash Buffer, mix gently by inversion, centrifuge at 1000 rpm for 1 min, and carefully remove the supernatant. Repeat at least twice. For gravity columns, wash and drain by gravity.
3.2.3 Binding
Add the prepared sample to the equilibrated resin. Seal the tube or column and mix by rotation. Incubate at room temperature for 30 min-2 h or at 2-8 °C for 2-4 h (longer if needed). Keep at 2-8 °C if the protein is unstable. Centrifuge at 1000 rpm for 1 min and retain the supernatant for analysis.
3.2.4 Washing
Wash with 5 CV Wash Buffer, mix gently, centrifuge at 1000 rpm for 1 min, and retain the supernatant. Repeat 3-5 times. For gravity columns, wash and drain by gravity.
3.2.5 Elution
Elute with 3-5 CV Elution Buffer Containing Maltose Monohydrate. Incubate at room temperature for 5 min. Centrifuge at 1000 rpm for 1 min or collect by gravity flow. Repeat 2-3 times. After elution, wash with 3 CV equilibration buffer, 5 CV purified water, and 2 CV storage solution. Store the resin at 2-8 °C.
4 Sample Detection and Resin Cleaning
4.1 SDS-PAGE
Analyze samples from the purification process (starting material, flow-through, wash, and elution fractions) by SDS-PAGE to evaluate purification efficiency.
4.2 Resin Cleaning
After eluting the target protein, the column is washed with pure water for 3-5 column volumes, then regenerated with 0.5 M NaOH for 3-5 column volumes. It is then washed with pure water until neutral, and stored in 20% ethanol or equilibrated with the binding buffer for the next purification. The recommended flow rate is 75-150 cm/h.
Matters needing attention
1. Do not freeze the Dextrin Resin.
2. Dextrin Resin is supplied in 1×PBS containing 20% ethanol, with a 1:1 ratio of settled resin to storage solution.
3. The resin must be thoroughly resuspended by repeated inversion before use to ensure uniform agarose bead suspension.
4. This product is for research use only.
5. For your safety and health, wear a lab coat and disposable gloves during all experiments.
