STarm Streptactin Beads 4FF
Strep-tag is a widely used affinity tag in protein purification systems. It consists of two types Strep-tag II and Twin Strep-tag II. Strep-tag II is a short peptide tag consisting of 8 amino acids (WSHPQFEK) that can be fused to proteins as an N-terminal or a C-terminal tag with minimal effect on recombinant proteins. The further improved Twin Strep-tag II is a sequence of two Strep-tag II sequences in a sequential order (linked by internal amino acids), and this tag is capable of gentle and rapid purification like Strep-tag II. The two tags are free to bind either of the ligands in Streptactin and STarm Streptactin. Tag/ligand binding depends on the desired binding strength and application. The affinity of these two tags to Streptaction and STarm Streptaction is in the μg/ml range, a high affinity that is not achieved by any other existing affinity labeling system. In addition, this flexibility of combining tags and ligands allows purification of recombinant proteins under physiological conditions.
Strep-tag labeling technology can be used to purify functional strep-tagged proteins from a variety of expression systems, including baculoviruses, mammalian cells, yeast, and bacteria. In general, both of these tags do not interfere with the folding or biological activity of the target proteins, do not react with heavy metal ions, do not have ion-exchange properties, and do not cause protein aggregation. Therefore, there is no need to remove Strep-tag II and Twin Strep -tag II after purification.
The ligand protein for STarm Streptactin Beads 4FF is Streptactin Mutant coupled to highly cross-linked 4% agarose microspheres. Low concentrations (50 μmol/L) of D-Biotin in the sample do not affect the binding of the target protein to STarm Streptactin Beads 4FF. The product can be regenerated with an equilibrium solution after use or cleaned with 10 mM NaOH. The characteristics are shown in Table 1.
Figure 1. Schematic diagram of Strep-tag Ⅱ and Twin Strep -tag Ⅱ combined with Streptaction
Table 1. Characteristics of PreCap STarm Streptactin
Item | Description |
Matrix | Highly cross-linked 4% agarose beads |
Ligand | Streptactin Mutant |
Capacity（/ml medium） | 4 mg twin Strep-tag II fusion proteins |
Particle Size (μm) | 45-165 μm |
Maximum Flow Rate | 300 cm/h |
Storage Buffer | 1×PBS containing 20% ethanol |
Storage Temperature | 2-8℃ |
Table 2. Chemical compatibilities for PreCap STarm Streptactin
Reagent | Contact time |
6 M Guanidine hydrochloride | 2 hours |
8 M Urea | |
2 M NaCl | |
50 mM DTT | 1 hour |
50 mM β-Mercaptoethanol | |
1 mM TCEP | |
0.1% SDS | |
2% Triton X-100 | |
2% Tween 20 | |
0.25 M imidazole | |
25% glycerol | |
1 M (NH4)2SO4 | |
0.1 M MgCl2 | |
0.1 M CaCl2 |
Figure 2. CIP Cleaning of STarm Streptactin Beads 4FF
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.
LB medium: 10 g/L peptone, 5 g/L yeast powder, 5 g/L NaCl
Antibiotic: 50 mg/ml Kana
Induction agent: 1 mol/L IPTG
2×SDS-PAGE Loading Buffer: 100 mM Tris-HCl, 20% glycerol, 4% SDS, 0.1% bromophenol blue, 200 mM DTT, pH 6.8
Binding/Wash buffer: 100 mM Tris-HCl, 150 mM NaCl, 1mM EDTA, pH 8.0 or PBS
Elution Buffer: 1-5 mM D-Biotin in binding buffer
Regeneration Buffer: 10 mM NaOH
2.2 Sample preparation
The sample should be adjusted to the composition of the binding buffer. This can be done either by diluting the sample with binding buffer or by buffer exchange. It is recommended to filter the sample solution by passing them through a 0.22 μm or 0.45 μm filter before use.
Streptactin Beads 4FF 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.
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, STarm Streptactin Beads 4FF is packed at a constant pressure of approximately 3bar(0.3MPa).If the packing equipment does not include a pressure gauge,use a packing flow velocity of approximately 400cm/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. Don′t 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.
2.3 Sample Purification
STarm Streptactin Beads 4FF binds Strep-Tag II in three general steps: Binding, Wash, and Elution (Figure 3).
Figure 3. Schematic diagram of STarm Streptactin Beads 4FF purification
1) Fill the syringe or pump tubing with distilled water. 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 stopper at the column outlet and connect the column to the chromatographic system.
2) Wash the column with 3-5 column volumes of distilled water.
3) Equilibrate the column with at least 5 column volumes Lysis Buffer.
4) Apply the pre-treated sample, using a Loop fitted to the connector or by pumping it onto the column.
5) Wash with Wash Buffer until the absorbance reaches the baseline or no material appears in the effluent (Generally at least 10-15 column volumes).
6) 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 target protein. Use UV absorbance, SDS-PAGE, or western blot.
2.5 Preservation
STarm Streptactin Beads 4FF should be regenerated once after each use to remove D-Biotin bound to the media to ensure consistent results, as specified:
In general, STarm Streptactin Beads 4FF 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 exceeds 3 bar | Filters are clogged | Clean or replace the filter. |
Column is clogged | Cleaning in place (part 3). | |
Filter the sample solution by passing them through a 0.22μm or 0.45 μm filter. | ||
Curve instability during sample purification | Air bubbles in the sample or buffer | Removal of air bubbles from samples or columns. Sample and buffer are degassed. |
Large temperature differences between the sample or buffer and the medium | Sample, buffer and medium are placed at the same temperature for purification. | |
No protein is eluted | Target proteins not expressed or expressed in low amounts | Optimize the expression of target proteins. |
Protein degradation or cleavage | Addition of appropriate amounts of protease inhibitors and protectants to the lysate. Purification at low temperature. | |
Strong binding of target proteins | Increase biotin elution concentration. | |
The elute is not pure | Protein degradation or cleavage | Addition of appropriate amounts of protease inhibitors and protectants to the lysate. Purification at low temperature. |
Contaminant proteins interact with target proteins | Add a low concentration of reducing agent to the buffer and sample lysate. Add final concentration of 0.1% Triton X-100 to the buffer. | |
Insufficient equilibration/wash operations | Increase the equilibrium liquid volume to ensure that the media is adequately equilibrated/washed, if the media is too dirty follow cleaning in place (part 3). |
Product | Cat. No. | Size |
STarm Streptactin Beads 4FF | SA092005 SA092025 SA092100 SA092500 SA09201L SA09210L | 5 ml 25 ml 100 ml 500 ml 1 L 10 L |
