Persefose CL-4B

Persefose CL-4B is a modified cross-linked agarose-based gel filtration SEC resin (cross-linking ratio 4%), which separates different molecules by their molecular weight and conformation differences. It can be used successfully for the separation and purification of a wide range of biological molecules, such as recombinant proteins, antibodies, nucleic acids, viruses and virus-like particles, and polysaccharides, as well as for QC molecular weight determination. It is particularly suitable for molecules that are not readily soluble in aqueous solution.

Method of use

Chromatographic conditions

Buffer selection. Consider the stability of the sample in the buffer. To avoid possible non-specific adsorption, use a salt-containing buffer rather than ultrapure or pure water.

Flow rate. Generally select a linear flow rate no higher than 30 cm/h, according to the height of the column bed.

Sample pretreatment. Filter the sample through a 0.45 µm microporous membrane before loading to prevent the sample from clogging the column.

Chromatography steps

1. Equilibration. Use the buffer to fully equilibrate the chromatography column until the pH and conductivity are stable and basically consistent with the equilibration buffer. This step usually requires 1–2 column bed volumes (CV).
2. Sample loading. The usual loading volume is 2%–5% of the column volume. The sample concentration should not be too high, to avoid overpressure or affecting the resolution.
3. Elution. Use buffer to elute, collecting the components that elute at different positions, usually over 1~1.5 CV.
4. Regeneration. Rinse the column with a high-salt buffer (such as 1M NaCl).
5. Re-equilibration. Re-equilibrate the column with buffer.

Cleaning and regeneration

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

For different types of impurities and contaminants, the recommended cleaning conditions are as follows:

• Strongly binding proteins: wash with 5 CV of 2M NaCl solution, or use a high salt buffer not lower than pH 3, such as 1M NaAc solution.
• Strongly hydrophobic proteins and precipitated proteins: first wash with 5 CV of 1M NaOH solution, then wash out the lye with 5–10 CV of ultrapure or pure water.
• Lipoproteins and lipids: first wash with 5 CV of 70% ethanol or 30% isopropanol, then rinse with 5–10 CV of ultrapure or pure water.

Degas 70% ethanol or 30% isopropanol before use. The flow rate should be no more than 30 cm/h during CIP. Use reverse cleaning where the clogging is severe. To reduce the microbial load, treat the chromatography resin with 0.5~1M NaOH solution for 15–30 minutes.

Storage

Keep the unopened chromatography resin in the original container and store 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

Since chromatography resin is difficult to degrade in nature, it is recommended that the waste chromatography resin is incinerated to protect the environment. For chromatography resin that has been in contact with biologically active samples such as viruses and blood, follow the 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

• 703-00025 — 25 ml
• 703-00100 — 100 ml
• 703-00500 — 500 ml
• 703-01000 — 1 L
• 703-05000 — 5 L
• 703-10000 — 10 L
• 703-20000 — 20 L