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Phenyl Crystarose 6FF(HS)（Hydrophobic Chromatography Resin）

classification：Agarose packing

classification：Hydrophobic chromatography

Phenyl Crystarose 6FF is a kind of Hydrophobic Interaction Chromatography HIC (Phenyl Crystarose)

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Phenyl Crystarose 6FF

I. Introduction

Phenyl Crystarose 6FF is a kind of Hydrophobic Interaction Chromatography HIC (Phenyl Crystarose), which is based on the principle that in a high concentration salt solution, the hydrophobic groups of proteins are adsorbed onto the hydrophobic groups of hydrophobic chromatography packing by hydrophobic adsorption, and then eluted by gradually decreasing the salt concentration. The protein is then eluted by gradually reducing the salt concentration, and the protein is eluted successively according to the strength of hydrophobicity. This simple and practical hydrophobic chromatographic packing is particularly suitable for the separation of samples containing biological macromolecules with high salt content, including peptides and nucleic acids and viruses. It can also be used for the separation and purification of some natural products and decolorization, etc.

The product will be our self-designed synthetic phenyl agarose gel, this hydrophobic chromatography media with high loading capacity, good physical and chemical stability, long service life, easy to operate. Good batch reproducibility, easy to amplify, so it is ideal for both R&D and production, fully compatible with imports.

Second, the affinity filler characteristics:

III. Scope of application

Penyl Crystarose 6FF is more hydrophobic and widely used for the separation and purification of biological macromolecules such as proteins, peptides, nucleic acids and viruses, and also for the separation and purification of some natural products.

IV. Application examples

Experiment name: Phenyl agarose gel FF for the separation of antibodies.

Experimental steps:

1, take the appropriate amount of Penyl Crystarose 6FF loading column, 1.6 × 20m, the volume of the column bed for 10ml

2、 Equilibrate 2-5 bed volumes with 1-2M ammonium sulfate buffer at a flow rate of 5ml/min

3、 Add the same concentration of ammonium sulfate and equilibrium buffer solution in the sample to maintain the same, filter with 0.45μm filter membrane, on the sample, the flow rate of 2ml/min

4、 Rinse 2-5 volumes of column bed with equilibrium buffer at a flow rate of 2ml/min

5、 Then elute with elution buffer at a flow rate of 2ml/min, collect the elution peak and purify the effect by SDS-PAGE.

6、 Wash 5 volumes of the column bed with pure water, then wash 3 volumes of the column bed with 20% ethanol flow at a flow rate of 5ml/min, and store the column in +4~8℃ environment.

7. The chromatogram is shown in Figure 1, and the SDS-PAGE results are shown in Figure 2.

Buffer composition:

Equilibrium buffer: 20mM PBS, 1M ammonium sulfate, pH7.0.

Elution buffer: 20 mM PBS, pH 7.0.

V. Precautions for application:

1) Chromatographic column loading

1. The temperature of all the materials to be used should be the same as the temperature of the chromatographic operation, and the liquid is better to do degassing.

2. Add 20% ethanol at the lower end of the column to remove the air in the column, close the column outlet, and keep a small amount of 20% ethanol in the column.

3、When pouring the gel into the column continuously, use the glass rod's to draw the flow close to the inner wall of the column to reduce the generation of air bubbles and let the

the packing material to settle naturally first.

4. Before loading the column, the packing material should be left at room temperature for at least 2-3 hours to avoid the generation of air bubbles due to temperature changes when loading the column.

2) Protein binding

By increasing the salt concentration, the binding force of protein and packing can be increased, thus achieving the purpose of improving the separation effect. Commonly used salts are ammonium sulfate, sodium chloride, sodium sulfate, potassium chloride, etc.

3) Elution of protein

1. If the protein binding force is strong and cannot be eluted with the usual buffer, some organic solvents can be added to the eluent liquid, such as ethylene glycol, glycerol, ethanol, etc.

Sixth, regeneration cleaning

Wash 5 column bed volumes with pure water, then wash 5 bed volumes with 0.5M NaOH respectively, then wash 3 column bed volumes with water, then wash 5 column bed volumes with 70% ethanol or 30% isopropanol flow, then 20% ethanol flow wash 3 column bed volumes. In the application, some substances bound on the column such as denatured proteins and lipids cannot be removed by the regeneration process. In this case, the column can be washed with 0.5-1% nonionic surfactant for 5 column bed volumes, and then the column can be washed with organic solvent as the regeneration method above, which is more effective. Organic solvent mixed with water is easy to produce bubbles, in order to avoid such a situation, the prepared organic solvent can be placed at room temperature overnight and then used, which can avoid bubbles into the column and cause the column can not be used properly.

VII. Storage

In 20% ethanol, stored at 4℃ for a long time.

Key words：

Phenyl Crystarose 6FF(HS)