This product can be used for the purification of Strep-tag II and Twin Strep-tag fusion proteins from any expression system, including baculovirus, mammalian cells, yeast, and bacteria. Strep-tag II is a short sequence of 8 amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that has a negligible impact on the recombinant protein, making tag removal unnecessary. The further improved Twin Strep-tag consists of two sequentially arranged Strep-tag II sequences (connected by a linker), enabling gentle and rapid purification just like the Strep-tag II. Streptavidin ST is one of the most stable proteins. Its conjugation to highly cross-linked 4% agarose microspheres allows for the affinity purification of fusion proteins under physiological conditions, ensuring protein bioactivity.
Aladdin Strep II Agarose Resin is stored in 1× PBS containing 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
| Highly cross-linked 4% agarose microspheres
|
Ligand
| Streptavidin ST
|
Particle Size Range
| 45~165 μm |
Binding Capacity
| 3–5 mg Twin-Strep-tag protein/mL resin
|
| Maximum Flow Rate | 300 cm/h |
Storage Conditions
| 1× PBS with 20% ethanol, 2–8°C
|
Shelf Life
| 2 years |
Protocol
1. Buffer Preparation
Water and buffers should be filtered through a 0.22 μm or 0.45 μm membrane before use.
Equilibration/Wash Buffer: 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0
or PBS: 20 mM sodium phosphate, 280 mM NaCl, 6 mM potassium chloride, pH 7.4
Elution Buffer: Equilibration buffer supplemented with 2.5 mM desthiobiotin
Regeneration Buffer: Equilibration buffer supplemented with 1 mM HABA (2-(4-hydroxyphenylazo)benzoic acid)
Note: If free biotin is present in the sample, dialyze prior to use to avoid affecting resin reusability.
2. Sample Preparation
Clarify samples by centrifugation or filtration (0.22/0.45 μm) before loading to reduce impurities, improve efficiency, and prevent column clogging.
3. Column Packing
3.1 Gravity Column Packing
(1) Select a suitable gravity column. Insert the bottom frit, rinse with purified water, and close the outlet.
(2) Resuspend the resin thoroughly. Transfer an appropriate volume of slurry to the column (settled resin volume is half of the slurry). Open the outlet to drain the storage solution.
(3) Rinse with purified water. Close the outlet after draining.
(4) Insert the top frit, ensuring no gaps and a horizontal position.
(5) Equilibrate with equilibration buffer or store with storage solution at 2–8°C.
3.2 Medium-Pressure Column Packing
For industrial-scale purification, medium-pressure columns are often used. Calculate the required resin volume using:
V = 1.15 × π × r² × h
V: Resin volume (mL)
1.15: Compression factor
r: Column radius (cm)
h: Packing height (cm)
Note: The slurry volume should be twice the settled resin volume.
(1) Rinse the column bottom frit and adapter with deionized water. Ensure no bubbles are trapped. Close the outlet and retain 1–2 cm of water.
(2) Resuspend the resin and pour the slurry continuously into the column along the wall to minimize bubbles.
(3) If using a reservoir, fill it with water immediately. Place the distributor on the slurry surface and connect to the pump, avoiding bubbles.
(4) Open the outlet and start the pump at a low flow rate, gradually increasing to the final packing flow rate. Avoid disturbing the bed. Use the pump’s maximum flow rate if needed. After stabilization, flush with 3 CV deionized water. Mark the bed height.
(5) Stop the pump and close the outlet.
(6) Remove the reservoir and place the distributor inside the column.
(7) Lower the distributor to the marked bed height. Allow buffer to enter the distributor and tighten the adapter.
(8) Connect the column to the pump or chromatography system for equilibration. Adjust the distributor if needed.
4. Sample Purification
4.1 Batch Purification
(1) Transfer resin to a centrifuge tube. Centrifuge at 1,000 rpm for 1 min and discard supernatant.
(2) Wash with 5 resin volumes of equilibration buffer. Repeat twice.
(3) Add sample. Incubate at 4°C with shaking for 2–4 h or at 37°C for 30 min–2 h.
(4) Centrifuge at 1,000 rpm for 1 min. Collect supernatant as flow-through for analysis.
(5) Wash with 5 resin volumes of wash buffer. Repeat 3–5 times. Change tubes if needed.
(6) Elute with 3–5 resin volumes of elution buffer. Incubate at RT for 5 min. Collect eluate. Repeat 2–3 times.
4.2 Gravity Column Purification
(1) Equilibrate the column with 5 CV of equilibration buffer. Repeat 2–3 times.
(2) Load sample. Allow at least 2 min residence time. Collect flow-through. Reload to enhance binding.
(3) Wash with 10–15 CV of wash buffer. Collect wash fractions.
(4) Elute with 5–10 CV of elution buffer. Collect fractions (1 CV per tube) for analysis.
4.3 Medium-Pressure Column Purification
(1) Prime the pump with deionized water. Connect the column to the system and tighten.
(2) Flush with 3–5 CV deionized water to remove storage buffer.
(3) Equilibrate with at least 5 CV of equilibration buffer.
(4) Load sample via pump or sample loop. Note: High viscosity increases backpressure. Do not exceed the binding capacity.
(5) Wash with wash buffer until UV absorption stabilizes (typically 10–15 CV).
(6) Elute with 5–10 CV of elution buffer. Collect eluate containing the target protein. After elution, wash with 5–10 CV equilibration buffer, 5–10 CV water, and 2 CV 20% ethanol. Store at 2–8°C.
5. SDS-PAGE Analysis
Analyze samples (flow-through, wash, elution, and crude sample) by SDS-PAGE to evaluate purification efficiency.
6. Resin Regeneration and Cleaning
Regeneration: Wash with 5 CV deionized water. Regenerate with 15 CV regeneration buffer (equilibration buffer + 1 mM HABA), followed by 30 CV equilibration buffer. Desthiobiotin is displaced by HABA, which turns red upon binding to Streptavidin ST. HABA is then removed by equilibration buffer, and the column is ready for reuse.
Storage: After regeneration and cleaning, store the resin in an equal volume of storage solution at 2–8°C to prevent bacterial contamination.
