This product utilizes the interaction between biotin and streptavidin ligands for the purification of biotin or biotinylated proteins, antibodies, and other biomolecules. The affinity between streptavidin and biotin is extremely strong, requiring denaturing conditions for elution. In contrast, streptavidin's affinity for iminobiotin is relatively weaker, allowing binding at pH 9.5–11.0 and elution at pH 4.0 without denaturants, thereby better preserving the activity of affinity conjugates. The streptavidin affinity chromatography medium is based on a highly cross-linked 6% agarose matrix, offering high flow rates and chemical stability, making it suitable for large-scale purification.
Aladdin Streptavidin 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 6% agarose microspheres
|
Ligand
| Streptavidin
|
Particle Size Range
| 45~165 μm |
Binding Capacity
| >200 nmol biotin/mL resin
|
Maximum Pressure
| 0.3 MPa, 3 bar
|
pH Stability Range
| 4-9 |
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.
For Biotin or Biotinylated Molecules
Equilibration/Wash Buffer: 20 mM NaH₂PO₄, 0.15 M NaCl, pH 7.4
Elution Buffer: 8 M guanidine hydrochloride, pH 1.5
For Iminobiotin-Tagged Molecules
Equilibration/Wash Buffer: 50 mM ammonium carbonate, 0.5 M NaCl, pH 10.0
Elution Buffer: 50 mM ammonium carbonate, 0.5 M NaCl, pH 4.0
2. Sample Preparation
Ensure the sample solution has appropriate ionic strength and pH before loading. Dilute serum samples, ascites, or cell culture supernatants with equilibration/wash buffer, or dialyze the sample against equilibration/wash buffer.
Clarify the sample by centrifugation or filtration (0.22/0.45 μm) 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 streptavidin 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
(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 the recommended pressure/flow is not achieved. After the bed stabilizes, 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.
6. Resin Cleaning
Clean the resin if performance declines due to non-specific binding or aggregation:
Precipitated/denatured material: Clean with 2 CV of 0.1 M NaOH, 6 M guanidine HCl, or 8 M urea, followed by 5 CV PBS, pH 7.4.
Hydrophobic adsorption: Clean with 3–4 CV of 70% ethanol or 2 CV of 1% Triton X-100, followed by 5 CV PBS, pH 7.4.
