This product is a chromatography separation medium formed by coupling a streptavidin mutant (ST2 ligand) to agarose gel. It is designed for the separation and purification of Strep-tag II or Twin-Strep-tag proteins. The Strep-tag II is a small 8-amino acid tag (WSHPQFEK) that generally does not affect the structure or function of the fused protein, making it widely used for the detection and purification of fusion proteins. The Twin-Strep-tag consists of two tandem Strep-tag II sequences and exhibits higher affinity for the ST2 ligand compared to the single Strep-tag II.
The ST2 ligand in this product shows further enhanced affinity for Strep-tag II and significantly reduced affinity for biotin compared to the previous ST ligand. This allows for stronger binding to Strep-tag fusion proteins, higher relative capture efficiency at low expression levels, and increased binding capacity and yield. For elution, biotin can be used instead of the more expensive desthiobiotin, making the process more economical. After use, the medium can be regenerated with 10–50 mM NaOH. The product offers high specificity for the Strep-tag, typically enabling high-purity protein samples in a single purification step.
Aladdin Strep II Agarose Resin (Biotin-Resistant) is stored in 20% ethanol, with a settled gel to storage solution ratio of 1:1. The product specification refers to the actual volume of the settled gel.
Parameter
| Value / Description
| Matrix
| 4% cross-linked agarose
| Ligand
| ST2 Ligand (streptavidin mutant), ≥5 mg/mL
| Particle Size Range ①
| 45~165 μm
| Average Particle Size
| ~90 μm
| Binding Capacity ②
| 8–10 mg Strep-tag II protein/mL resin
| Recommended Operating Flow Rate
| 60~150 cm/h
| Maximum Flow Rate & Pressure ③
| 900 cm/h，0.3 MPa
| Operating pH Range
| 6–10 (recommended), 10–50 mM NaOH (CIP)
| Chemical Stability
| Stable in common aqueous buffers, 8 M urea, etc.
| Storage Conditions
| 20% ethanol, 2–8°C
| Shelf Life
| 3 years |
|
Notes:
① Over 90% of the beads fall within this size range.
② Binding capacity measured as pure protein yield (enhanced green fluorescent protein with Strep-tag II) per mL resin; comparable to DBC₁₀%.
③ Maximum tested flow rate at 10 cm column height.
Protocol
1. Column Packing
The following procedure describes column packing when connected to a chromatography system:
(1) Equilibrate all materials to the operating temperature. Degas liquids if possible.
(2) Resin Quantity Calculation:
Settled resin volume = Column volume × Compression factor (1.15 for this resin).
(3) Resin Preparation: Resuspend the resin slurry thoroughly. Measure the required volume, remove storage solution by filtration, and wash 3 times with ~3 resin volumes of purified water.
(4) Packing Slurry Preparation: Transfer resin to a suitable container and add packing solution to achieve a 50–75% (v/v) slurry. Mix well before use.
(5) Pre-packing Setup: Fill the bottom of the clean column with packing solution to remove air from the bottom frit and outlet. Retain a small amount of liquid, tighten the bottom end fitting, and ensure the column is vertical.
(6) Packing: Pour the well-mixed slurry into the column in one slow, continuous motion (use a packing reservoir if needed). After adding all resin, fill the reservoir with packing solution, attach the top lid, and connect the column to the system.
(7) Compression: Allow the resin to settle naturally (or use a low flow rate of 50–150 cm/h). Apply a high flow rate (600 cm/h; ensure pressure ≤0.3 MPa) until the interface is stable. Mark the stable height, stop the pump, and lower the adapter to the position corresponding to the compression factor (1.15). Tighten the adapter and equilibrate the column at a high flow rate.
Packing Condition
| Strep II Agarose Resin (Biotin-Resistant)
| Compression Factor
| 1.15
| Packing Flow Rate
| 600 cm/h |
|
2. Column Efficiency Testing
After packing, assess column efficiency using HETP (Height Equivalent to a Theoretical Plate) and As (Asymmetry Factor) with acetone or NaCl as tracers:
Tracer
| 1.0% Acetone
| 0.8~1.0 M NaCl
| Sample Volume
| 1.0% CV
| 1.0% CV
| Mobile Phase
| Pure Water
| 0.4 M NaCl
| Flow Rate
| 30 cm/h
| 30 cm/h
| Detector
| UV-280 nm
| Conductivity |
|
Calculate HETP, N, and As using:
HETP = L / N
N = 5.54 × (Vʀ / Wₕ)²
As = a / b
HETP should be <3× the average particle diameter (HETP/D₅₀ < 3), and As should be 0.8–1.5.
3. Equilibration and Loading
Recommended Buffer A (Binding/Equilibration Buffer): 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0.
Clarify the sample and dilute/exchange it into Buffer A.
Equilibrate with 5 CV of Buffer A at ~150 cm/h.
Load sample at 50–150 cm/h; lower flow rates may improve binding capacity.
4. Washing
5. Elution
Recommended Buffer B (Elution Buffer): 50 mM D-biotin, 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0.
Elute with 6–10 CV of Buffer B at ~150 cm/h.
6. Regeneration
7. Storage
ZJ25F1230742