Dextrin Beads 6FF
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.
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.
Dextrin Beads 6FF is easy to pack and use, and its high flow properties make it excellent for industrial scaling-up. The method of
packing the column is described below.
2) Close the column outlet leaving the net covered with packing buffer.
3) Resuspend the beads stored in its container by shaking (avoid stirring the sedimented medium). Pouring the slurry down a glass rod held against the column wall will minimize the introduction of air bubbles.
If using a packing reservoir, immediately fill the remainder of the column and reservoir with packing buffer. Mount the adapter or lid of the packing reservoir and connect the column to a pump. Avoid trapping air bubbles under the adapter or in the inlet tubing.
4) Open the bottom outlet of the column and set the pump to run at the desired flow velocity. Ideally, Dextrin Beads 6FF is packed at a constant pressure of approximately 3 bar (0.3 MPa). If the packing equipment does not include a pressure gauge, use a packing flow velocity of approximately 400 cm/h (10 cm bed height, 25℃, low viscosity buffer).If the recommended pressure or flow velocity can not be
obtained, use the maximum flow velocity the pump can deliver. This should also give a reasonable well-packed bed. Do not exceed 75% of the packing flow velocity in subsequent chromatographic procedures.
5) When the bed has stabilized, close the bottom outlet and stop the pump.
If using a packing reservoir, disconnect the reservoir and fit the adapter to the column. If using the column, carefully place the top filter on top of the bed before fitting the adapter.
6) With the adapter inlet disconnected, push the adapter down, approximately 2 mm into the bed, allowing the packing solution to flush the adapter inlet.
7) Connect the pump, open the bottom outlet and continue packing. The bed will be further compressed at this point and a space will be formed between the bed surface and the adapter.
8) Close the bottom outlet. Disconnect the column inlet and lower the adapter approximately 2 mm into the bed. Connect the pump. The column is now ready to use.
2.4 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.
2.5 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 |
SA026005 SA026025 SA026100 SA026500 SA02601L SA02610L |
5 ml 25 ml 100 ml 500 ml 1 L 10 L |
