Octyl-Sepharose 4FF

Description:

Separation reagent, chromatographic medium. Particle size: 45-165μm; Maximum flow rate: 600cm/h; Load per milliliter: 5 micromoles of n-butyryl n-Octyl; Particle size (μL) : 45-165; Medium hydrophobic, suitable for the separation and purification of various proteins.

Product Introduction

Octyl-agarose gel 4FF is a medium hydrophobic medium formed by binding octyl-agarose gel to achieve purification and separation of target products by hydrophobic action. Suitable for the separation and purification of various proteins. It has the characteristics of fast flow rate, easy to use and wide application.

Product parameters

Substrate: 6% crosslinked agarose gel

Ligand: n-butanyl n-Octyl

Ligand density: 5μmol/mL

Particle size: 45-165μm

Maximum flow rate: 600cm/h

Working pH: 3-13

Operating temperature: room temperature

Instructions for use:

Octyl-agarose gel 4FF is a medium hydrophobic chromatography medium suitable for the separation and purification of various proteins.

How to use

1. Install the column

(1) Bring all reagents and fillers to room temperature. Prepare the initial buffer (balance) and buffer.

(2) Take the amount of gel required according to the size of the column, wash off 20% ethanol, drain it, and prepare a homogenate with the initial buffer (according to the gel: buffer =3:1 ratio).

(3) Wet the inside of the column and the bottom of the column with water or buffer and keep a small section of liquid (the liquid level is slightly higher than the filter membrane), be sure to make the bottom end free of bubbles.

(4) Use a glass rod to guide the homogenate along the inner wall of the column into the column at one time, taking care not to use bubbles. Open the liquid outlet of the column, make the gel settle freely in the column, and connect the top of the column.

(5) Open the peristaltic pump, so that the buffer flow rate of 1.33 times the flow rate of use, so that the column bed is stable. Balance the column with a buffer of 2-3 times the column volume.

Step 2 Balance

Let the equilibrium buffer flow through the column at a constant flow rate until the effluent conductance and pH are unchanged. The equilibrium buffer is generally a buffer of high salt concentration, such as: 0.02-0.05mol/L PBS plus 1-2.5mol/L(NH4)2SO4 and so on.

3. Sample delivery

(6) The sample is prepared with equilibrium liquid, and the turbid sample should be centrifuged and filtered on the column;

(7) In general, the target product is combined on the column, the impurities are washed with a balanced lotion, and then an eluent is selected to wash the target product.

(8) The degree of adsorption of the sample components depends on the hydrophobic properties of the sample, the ionic strength and temperature of the mobile phase. The higher the temperature, the higher the salt concentration or the stronger the hydrophobicity of the sample component, the stronger the adsorption of the component.

Step 4 Elution

The hydrophobic medium can be elution with reduced salt concentration. Elution can be enhanced by adding surfactants or organic solvents. The most commonly used eluent is a low salt concentration buffer, such as: 0.02-0.05mol/L PBS.

Step 5 Regenerate

Generally washed with a buffer of low salt concentration, washed more than 10 times the column volume, and then washed with a balance solution of binding protein to balance, can be used again. If deactivated proteins or lipids cannot be washed off during regeneration, they can be removed by in-place cleaning (CIP).

6. Clean while in position

(1) Proteins bound by ionic bonds can be removed with 2M NaCl.

(2) For precipitated proteins, proteins or lipids bound by hydrophobic action, 1M NaOH can be removed.

(3) For strongly hydrophobic binding proteins, lipids, etc., can be cleaned with 70% ethanol or 30% isopropyl alcohol of 4-10 times the column volume, but pay attention to the gradual increase in the concentration of organic solvents in a gradient manner, otherwise it is easy to produce bubbles.

After cleaning, balance the column with at least 3 times the column volume buffer.

7. Remove from heat source

Clean the column with 0.5M of sodium hydroxide for 5-6 hours or 0.1M of sodium hydroxide for 24 hours. Or remove with the following steps:

(1) Twice the column volume of 70% ethanol;

(2) Twice the column volume 50Mm Tris-HcL Ph7.5

(3) 1 column volume 4M urea

(4) Tris buffer 3 times the column volume +0.1M NaCl;

The above buffers are prepared in double steaming water without heat source.

Step 8 Disinfect

Wash 8-10 times the column volume with 0.5-1.0M NaOH at room temperature to balance the column with the initial buffer.

Precautions

(1) Sealed storage at 4℃-28℃(storage solution is 20% ethanol + 0.1M sodium acetate), ventilated, dry place, can not be frozen. The used columns are stored at 4℃(20% ethanol).

(2) When installing the column, using and preserving the column, it is necessary to avoid the column drying and air bubbles entering.

Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.

Remark：These protocols are for reference only. Solarbio does not independently validate these methods.

Note：

1. The products are all for scientific research use only. Do not use it for medical, clinical diagnosis or treatment, food and cosmetics, etc. Do not store them in ordinary residential areas.

2. For your safety and health, please wear laboratory clothes, disposable gloves and masks.

3. The experimental results may be affected by many factors, after-sale service is limited to the product itself and does not involve other compensation.