PlasmidCap Persefose HP

PlasmidCap Persefose HP is a thiophilic affinity resin made by fixing the sulfur-containing ligand 2-mercaptopyridine to high-resolution cross-linked agarose. Thiophilic affinity uses the interaction between electron donors and electron acceptors to separate and purify biomolecules; the interaction is strengthened in a high-salt environment and weakened in a low-salt environment. The optimised ligand density gives an appropriate affinity for supercoiled DNA, and the fine microspheres improve the load of higher-molecular-weight supercoiled DNA.

Method of use

Chromatographic conditions

Buffer selection. Equilibration, binding and washing buffer: 2.0M (NH₄)₂SO₄, 10 mM EDTA, 0.1M Tris, pH 7.5. Elution buffer: 1.7M (NH₄)₂SO₄, 10 mM EDTA, 0.3M NaCl, 0.1M Tris, pH 7.5.

Flow rate. Select a linear flow rate of 50–120 cm/h according to the column bed height.

Sample pretreatment. Filter the sample through a 0.45 µm microporous membrane before loading to prevent the column from clogging. Adjust the pH and conductivity of the sample to be consistent with the equilibration buffer, using dilution, ultrafiltration or desalting.

Chromatography steps

1. Equilibration. Use equilibration buffer to fully equilibrate the column until the pH and conductivity are stable and consistent with the equilibration buffer. This step usually requires 3–5 column bed volumes (CV).
2. Sample loading. Determine the loading volume and loading amount from the binding capacity measured in a small-scale test.
3. Washing. Use equilibration buffer or another suitable buffer to wash the column until the UV signal stabilises and returns to baseline.
4. Elution. Apply the elution buffer.
5. Regeneration. Wash with 3 CV of ultrapure or pure water, then clean with 3 CV of 0.5M NaOH followed by 3 CV of ultrapure or pure water.
6. Re-equilibration. Re-equilibrate the column with equilibration buffer.

Cleaning and regeneration

Contaminants such as lipids, endotoxins and proteins accumulate on the column as the number of uses increases. Regular cleaning-in-place (CIP) is essential to keep the column in stable working condition. Determine the frequency of CIP according to the degree of contamination. Where contamination is considerable, CIP after each use is recommended to ensure repeatability of results and to prolong the working life of the resin.

Recommended cleaning conditions by contaminant type:

• Denatured or precipitated proteins: wash with 5 CV of 0.5M NaOH solution, then with 5–10 CV of equilibration buffer.
• Lipoproteins and lipids: wash with 2–4 CV of 20 mM PB with 30% isopropanol, pH 7.5, then rinse with 5–10 CV of ultrapure or pure water.

Degas 30% isopropanol before use. Use a flow rate of 30–60 cm/h during CIP. Use reverse cleaning where clogging is severe.

To reduce microbial load, treat the resin with 70% ethanol for more than 12 hours.

Storage

Keep unopened resin in the original container at 4–30°C in a well-ventilated, dry and clean place. Do not freeze. Wash the used column with 2–3 CV of 20% ethanol solution and store at 2–8°C.

Destruction and recycling

Chromatography resin is difficult to degrade in nature, so incineration of waste resin is recommended. For resin that has been in contact with biologically active samples such as viruses and blood, follow local biosafety requirements before destroying or disposing of it.

Packing method

Detailed information on resin packaging is available on request. Please contact your local distributor.

Ordering information

• 181-00025 — 25 ml
• 181-00100 — 100 ml
• 181-00500 — 500 ml
• 181-01000 — 1 L
• 181-05000 — 5 L
• 181-10000 — 10 L
• 181-20000 — 20 L