Q-Sepharose Fast Flow

Introduction

Q-agarose gel FF is a kind of strong anion exchange medium formed by bonding trimethylamine alkyl quaternary ammonium groups on agarose microspheres with high flow rate. It is widely used for ion exchange preparation of downstream proteins, nucleic acids and peptides in biopharmaceuticals and bioengineering.

This product is white spherical gel, no smell, tasteless, no visible impurities.

Two, affinity packing characteristics

* Detection conditions: chromatographic column 10mm×300mm * column bed height 15cm, 25℃, mobile phase 0.1mol/LNaCl.

Scope of application

This product has the characteristics of high chemical stability, high flow rate, high load, good mechanical properties, can be cleaned in place, multiple reuse, etc., low non-specific adsorption, high recovery rate, suitable for industrial scale production, suitable for the separation and purification of biological macromolecules that can form negative ions in the pH working range. It is widely used for ion exchange preparation of downstream proteins, nucleic acids and peptides in biopharmaceuticals and bioengineering.

Four, operation instructions

1 column

(1) Allow all materials and reagents to reach room temperature. Prepare the initial buffer (equilibrium) and eluting buffer.

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

(3) Wet the inside of the column and the bottom of the column with water or buffer and maintain a small level (the level of the liquid is slightly higher than the filter), make sure that the bottom is 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 produce bubbles. Open the liquid outlet of the column, make the gel settle freely in the column, and connect the top of the column.

(5) Turn on the peristaltic pump and let the buffer flow at 1.33 times the flow rate when used to make the column bed stable. Balance the column with a buffer of 2 to 3 times the column volume.

2 Balance

Let the equilibrium buffer flow through the column at a constant flow rate until the effluent conductance and pH are unchanged. Equilibrium solution is a low concentration buffer solution, such as Tris, PBS, etc.

3 Sample

(1) The sample is prepared with a balanced solution, and the turbid sample is centrifuged and filtered before being fed. The salt concentration is too large sample after treatment.

(2) Generally, the target product is combined on the column, the impurities are washed with the balance solution, and then an eluent is selected to wash the target product.

(3) The degree of adsorption of sample components by the medium depends on the charged properties of the sample, the ionic strength of the mobile phase, and the pH value. With small salt concentration, the medium adsorbed the sample components more firmly. When using DEAE media, the recommended pH value is 1 unit less than the isoelectric point of the target product.

4 Elution

Q agarose gel FF medium can be eluted by increasing the salt concentration or decreasing the pH value, which is commonly used to increase the salt concentration.

5 regeneration

Generally wash with a buffer of high salt concentration (containing 1 to 2mol/L NaCl) or reduce the pH to wash more than 10 times the column volume, and then wash 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 In-place cleaning

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

(2) 1M NaOH can be used to remove precipitated proteins and hydrophobic bound proteins or lipids.

(3) For strongly hydrophobic bound proteins, lipids, etc., clean with 70% ethanol or 30% isopropyl alcohol of 4 to 10 times the column volume, but pay attention to the concentration of organic solvents gradually increase in a gradient way, otherwise it is easy to produce bubbles.

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

7 Attention

When installing the column, using and saving the column, it is necessary to avoid the column drying and air bubbles entering.

In the process of use, can not use strong acid, such as the use of pickling, should use a concentration of less than 0.1M glacial acetic acid.

8 Remove heat source

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

(1) 70% ethanol twice the column volume;

(2) Twice the column volume 50mM Tris-HCl pH7.5;

(3) 1 column volume 4M urea;

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

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

9 Disinfection

Wash 8-10 times the column volume with 0.5~1M NaOH at room temperature and balance the column with the initial buffer.

10 Sterilization

Place the medium in an autoclave at 120℃for 30 minutes.

Five, precautions

The product should be stored in a sealed, ventilated, dry and clean place at 4℃~25℃(the storage solution is 20% ethanol). It cannot be frozen. The used columns are stored at 4℃(20% ethanol). Avoid contact with oxidants; Avoid prolonged exposure in pH<4 environments (one week,20℃).

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.