Dextrin Beads 6FF Prepacked Column
Dextrin Beads 6FF is a chromatography medium for the isolation of proteins fused to maltose binding protein (MBP-tagged protein).The characteristics of Dextrin Beads 6FF are summarized in table 1. MBP tag often gives increased expression levels and higher solubility of the target protein. Proper folding of the protein has also been shown to be promoted by the MBP tag. Dextrin Beads 6FF can purify MBP fusion proteins in one step. The fusion proteins can be eluted gently with 10 mM maltose to protect the activity of fusion proteins.
Dextrin Beads 6FF Prepacked Column is one of a range of prepacked, ready-to-use columns for affinity chromatography. It is packed with 1 ml and 5 ml of Dextrin Beads 6FF. Five different packing sizes are available. Dextrin Beads 6FF Prepacked Column has the standard interface and can be adapted to all kinds of chromatography system, such as KTA. It is fast, simple and easy operation.
Table 1. Characteristics of Dextrin Beads 6FF
Item | Description |
Matrix | Highly cross-linked 6% agarose |
Ligand | Dextrin |
Capacity （/ml medium） | >10 mg MBP tagged protein (80 kDa) |
Particle size (μm) | 45-165 |
Maxi pressure | 0.3 MPa, 3 bar |
pH stability | 3-12 |
Storage buffer | 1×PBS containing 20% ethanol |
Storage | 2-8℃ |
2.1 Buffer Preparation
Water and chemicals used for buffer preparation should be high purity. It is recommended to filter the buffers by passing them through a 0.22 μm or 0.45 μm filter before use.
Binding /Wash Buffer: 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH7.4
Elution Buffer: 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 10 mM maltose, pH7.4
Note: 1 mM DTT or 10 mM β-mercaptoethanol can be included in the binding /wash buffer or elution buffer
2.2 Sample Preparation
It is recommended to filter the sample solution by passing them through a 0.22 μm or 0.45 μm filter before use.
2.3 Sample Purification
1) Fill the syringe or pump tubing with binding buffer. Remove the stopper and connect the column to the syringe (with the provided connector), or pump tubing, “drop to drop” to avoid introducing air into the column. Remove the snap-off end at the column outlet.
2) Wash the column with 10 column volumes of binding buffer.
3) Apply the sample, using a syringe fitted to the connector or by pumping it onto the column.
4) Wash with 5 to 10 column volumes of binding buffer or until no material appears in the effluent.
5) Elute with 5 column volumes of elution buffer. Other volumes may be required if the interaction is difficult to break.
2.4 Analysis
Identify the fractions containing the MBP-tagged protein. Use UV absorbance, SDS-PAGE, or western blot.
In general, Dextrin Beads 6FF is well suited for reuse several times. When reduced performance or an increase in back-pressure are
noted, you need to clean the medium with the solutions as follows
Problem | Probable Cause | Solution |
Back pressure is too high | Column is clogged | Cleaning in place(part 3) |
Sample solution contains precipitate | Filter the sample solution by passing them through a 0.22 μm or 0.45 μm filter. | |
No binding | Expression of target protein is very low | Check expression level of protein by estimating the amount in the extract, flow through, elute fraction and pellet upon centrifugation. Or apply large sample volume. |
There are some interference factors in the sample or buffer. | Sample dialysis or diluted with binding buffer. | |
Amylase produced by cells affected the protein combined with the medium. | Inhibit the expression of amylase by adding glucose to the culture medium. | |
Contact time is too short. | The sample and the medium was incubated for 2 hours at RT or longer. | |
The elute is not pure | Protein degradation | Add some protease inhibitors, such as PMSF, EDTA. |
Wash is not enough | Increase the volume of Wash Buffer. |
Product | Cat. No. | Size |
Dextrin Beads 6FF Prepacked Column | SA026C11 SA026C51 SA026C15 SA026C55 SA026CS | 1x1 ml 5x1 ml 1x5 ml 5x5 ml 3x1 ml+1X5 ml |
