BioReagent, 50% v/v BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
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Cited in 0 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
Lysine Affinity Resin 4FF is a medium designed for the purification of plasminogen activators, plasminogen, and ribosomal ribonucleic acid. Lysine is a basic amino acid and is an analogue of ε‑aminocaproic acid, which is an inhibitor of plasminogen activator. Lysine Affinity Resin 4FF consists of lysine coupled to highly cross‑linked 4% agarose beads, allowing purification of target proteins at relatively high flow rates. The specific performance characteristics are listed in Table 1.
Storage note:
Aladdin Lysine Affinity Resin 4FF is supplied in 1× PBS containing 20% ethanol. The volume ratio of gel to preservative solution is 1:1. The product specification refers to the actual settled gel volume.
Table 1: Performance specifications of Lysine Affinity Resin 4FF
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2. Purification Protocol
2.1 Buffer Preparation
You may use the recommended buffers below or prepare your own buffer systems according to your preferences. The basic principle is low‑imidazole loading and high‑imidazole elution. It is recommended to filter all buffers through a 0.22 μm or 0.45 μm membrane before use.
Table 2: Buffer formulations for soluble His‑tagged protein purification
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2. Purification Protocol
2.1 Preparation of buffers
It is recommended to filter all water and buffers through a 0.22 μm or 0.45 μm membrane before use.
Equilibration buffer: 50 mM phosphate buffer, pH 7.5
Wash buffer: 50 mM phosphate buffer, pH 7.5
Elution buffer: 50 mM phosphate buffer containing 1–2 M NaCl, pH 7.5
Competitive elution buffer: 0.2 M ε‑aminocaproic acid
2.2 Sample preparation
Prior to loading, it is recommended to centrifuge the sample or filter it through a 0.22 μm or 0.45 μm membrane to reduce particulates, improve purification efficiency, and prevent column clogging.
2.3 Packing of Lysine Affinity Resin 4FF
2.3.1 Packing a gravity column
1.Take a gravity column of appropriate size, insert the lower frit, rinse the column tube and frit with an appropriate amount of pure water, and close the bottom outlet.
2.Resuspend the Lysine Affinity Resin 4FF thoroughly, pipette an appropriate volume of the slurry into the gravity column (the actual medium volume is half of the slurry volume), then open the bottom outlet to let the preservative solution drain by gravity.
3.Add an appropriate amount of pure water to rinse the medium. After the liquid has drained by gravity, close the bottom outlet.
4.Insert the pre‑rinsed upper frit, ensuring that there is no air gap between the frit and the resin and that the frit is level.
5.The packed gravity column can be equilibrated directly with equilibration buffer. If not used immediately, add preservative solution and store at 2–8°C.
2.3.2 Packing a medium‑pressure chromatography column
Lysine Affinity Resin 4FF is widely used in industrial purification, so it is often packed into various medium‑pressure chromatography columns. The following method describes the packing procedure. Before packing, calculate the column bottom area based on the column diameter, and then calculate the required medium volume according to the desired bed height using the formula:
V = 1.15 × π × r² × h
(V = required medium volume in mL; 1.15 = compression factor; r = column radius in cm; h = packing height in cm)
Note: The volume of slurry to be taken should be twice the required medium volume, because the medium occupies only half of the slurry volume; the other half is the preservative solution.
1.Rinse the column bottom frit and adapters with deionised water, ensuring that no air bubbles remain on the bottom frit. Close the column bottom outlet and leave 1–2 cm of deionised water above the bottom frit.
2.Resuspend the medium and carefully pour the slurry continuously into the column. Pouring the slurry along the column wall with a glass rod can help reduce air bubbles.
3.If a reservoir is used, immediately fill the column and the reservoir with water. Place the sample distributor onto the slurry surface and connect it to the pump, avoiding air bubbles in the distributor or tubing.
4.Open the column bottom outlet and start the pump at the set flow rate. Initially, allow buffer to flow slowly through the column, then gradually increase to the final flow rate. This prevents hydraulic shocks and uneven bed formation. If the recommended pressure or flow rate cannot be reached, you may use the maximum flow rate of your pump; this can still give good packing results. (Note: In subsequent chromatographic runs, do not exceed 75% of the maximum packing flow rate.) Once the bed height is stable, wash with at least 3 column volumes of deionised water at the final packing flow rate. Mark the bed height.
5.Stop the pump and close the column outlet.
6.If a reservoir was used, remove it and place the distributor into the column.
7.Push the distributor down to the marked bed height. Allow packing fluid to enter the distributor, then lock the distributor adapter.
8.Connect the packed column to a pump or chromatography system and start equilibration. Adjust the distributor if necessary.
2.4 Sample purification
2.4.1 Batch incubation purification
1.Depending on the sample volume, take an appropriate amount of Lysine Affinity Resin 4FF into a centrifuge tube, centrifuge at 1000 rpm for 1 min, and discard the supernatant. Alternatively, the medium can be placed in a gravity column and the preservative solution drained by gravity.
2.Add 5 volumes of equilibration buffer (relative to the medium volume) to wash the medium. Centrifuge at 1000 rpm for 1 min and discard the supernatant; if using a gravity column, simply wash and allow the buffer to drain by gravity. Repeat this step at least twice.
3.Add the sample, seal the tube or column, and incubate with shaking at 4°C for 2–4 h or at 37°C for 30 min–2 h.
4.After incubation, centrifuge at 1000 rpm for 1 min and discard the supernatant, or filter to collect the medium. Retain the supernatant as the flow‑through fraction for electrophoresis analysis.
5.Wash the medium with 5 volumes of wash buffer, centrifuge at 1000 rpm for 1 min (or filter through the gravity column), and remove the supernatant (taking care not to aspirate the medium). Repeat this 3–5 times, and it is recommended to change to a new centrifuge tube in between.
6.Add 3–5 column volumes of elution buffer and incubate at room temperature for 5 min. Centrifuge at 1000 rpm for 1 min or collect the eluate through the gravity column. The elution step can be repeated 2–3 times.
Gravity column purification
1.Equilibrate the packed Lysine Affinity Resin 4FF gravity column with 5 column volumes of equilibration buffer to bring the resin into the same buffer system as the target protein. Repeat this 2–3 times.
2.Apply the sample to the equilibrated gravity column. The sample should have a residence time of at least 2 minutes to ensure sufficient contact between the sample and the medium. Collect the flow‑through. You may re‑load the flow‑through to increase binding efficiency.
3.Wash with 10–15 column volumes of wash buffer to remove non‑specifically bound contaminants, and collect the wash fractions.
4.Elute with 5–10 column volumes of elution buffer, collecting fractions in volumes of one column volume each. Assay each fraction individually; this ensures complete elution of all bound target protein and also allows collection of high‑purity and concentrated protein.
Medium‑pressure chromatography column purification
After packing, Lysine Affinity Resin 4FF can be used with a variety of conventional low‑ and medium‑pressure chromatography systems.
1.Fill the pump tubing with deionised water. Remove the top stopper, connect the column to the chromatography system, open the bottom outlet, mount the packed column, and tighten the connections.
2.Rinse out the storage buffer with 3–5 column volumes of deionised water.
3.Equilibrate the column with at least 5 column volumes of equilibration buffer.
4.Load the sample using the pump or a sample loop. Note: Increased sample viscosity can cause high backpressure even with small loading volumes. Do not exceed the binding capacity of the column. Large sample volumes may also generate high backpressure and make the injector more difficult to use.
5.Wash the column with wash buffer until the UV absorbance returns to a stable baseline (generally at least 10–15 column volumes).
6.Elute with 5–10 column volumes of elution buffer, and collect the eluate, which contains the target protein.
After elution is complete, wash the column with 5–10 column volumes of equilibration buffer, followed by 5–10 column volumes of pure water, and then with 2 column volumes of 20% ethanol. Store the column at 2–8°C.
2.5 SDS‑PAGE analysis
Analyse the purified product fractions (including flow‑through, wash, and elution fractions) along with the original sample by SDS‑PAGE to assess the purification performance.
3. Resin Regeneration and Cleaning
3.1 Resin regeneration
Wash with 3 column volumes of 0.1 M Tris‑HCl, 0.5 M NaCl, pH 8.5, followed by 3 column volumes of 0.1 M sodium acetate, 0.5 M NaCl, pH 4.5. Repeat this cycle 3 times.
Equilibrate with 5 column volumes of equilibration buffer.
After purification of plasminogen, the resin can be cleaned with 5 column volumes of binding buffer containing 0.2 M ε‑aminocaproic acid and 1 M NaCl.
After nucleic acid purification, the resin can be washed with 5 column volumes of binding buffer containing 2 M NaCl.
3.2 Cleaning of stubborn contaminants
Denatured proteins and lipids that are not eluted can be removed by washing with 0.1% Triton X‑100 at 37°C. Then re‑equilibrate with 5 column volumes of binding buffer.
3.3 Resin storage
The resin can be stored in 1× PBS containing 20% ethanol at 2–8°C.
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