rProtein A/G Beads 4FF Prepacked Column

rProtein A/G Beads 4FF Prepacked Column                 

1. Product Description

rProtein A/G Beads 4FF is an affinity chromatography medium designed for the purification of monoclonal antibody and polyclonal antibody at both laboratory and process scale. The rProtein A/G is coupled to highly cross-linked 4% agarose beads by a technique which generates a stable thioether linkage between rProtein A/G and the agarose beads. The coupling technique is optimized to give a high binding capacity for IgG.This binding capacity,together with the excellent kinetic and flow properties of the highly cross-linked beads.rProtein A/G Beads 4FF can be used for antibody purification, immuneprecipitation and Co- immuneprecipitation.

The characteristics of rProtein A/G Beads 4FF are summarized in Table 1.

rProtein A/G Beads 4FF Prepacked Column is a prepacked ready to use column for purification of monoclonal and polyclonal antibodies. rProtein A/G Beads 4FF Prepacked Column 1ml and 5ml columns are packed with 1ml and 5ml of rProtein A/G Beads 4FF. rProtein A/G Beads 4FF Prepacked Column can be operated with a peristaltic pump or liquid chromatography systems, such as ÄKTA.  It is fast, simple and easy operation.

Table 1. Characteristics of rProtein A/G Beads 4FF

Table 2. Relative binding strengths of antibodies from various species to protein G protein A and protein A/G as measured in a competitive ELISA test. 

++++=strong binding; ++= medium binding; —=weak binding or no binding

2. Purification Procedure

2.1 Buffer Preparation

Water and chemicals used for buffer preparation should be of high purity. It is recommended to filter the buffers by passing them through a 0.22 μm or 0.45 μm filter before use.

Binding/Wash Buffer: 20 mM Na₂HPO₄, 0.15 M NaCl ,pH 7.0

Elution Buffer: 0.1 M glycine, pH 3.0

Neutralization Buffer: 1 M Tris-HCl, pH 8.5  

2.2 Sample Preparation

To ensure that proper ionic strength and pH are maintained for optimal binding, it is necessary to dilute serum samples, ascite fluid or cell culture supernatant at least 1:1 with Binding/Wash Buffer. Alternatively, the sample may be dialyzed overnight against Binding/Wash Buffer.

It is recommended to filter the sample solution by passing them through a 0.22μm or 0.45 μm filter before use.

2.3 Sample Purification

1) Prepare collection tubes by adding 60 to 200 µl 1 M Tris-HCl, pH8.5 per ml of fraction to be collected.

2) Fill the syringe or pump tubing with binding buffer. Remove the stopper and connect the column to the syringe (with the provided connector), or pump tubing, “drop to drop” to avoid introducing air into the column. Remove the snap-off end at the column outlet.

3) Wash the column with 10 column volumes of binding buffer at 1 ml/min or 5 ml/min for 1 ml and 5 ml column respectively.

4) Apply the sample, using a syringe fitted to the connector or by pumping it onto the column.

5) Wash with 5 to 10 column volumes of binding buffer or until no material appears in the effluent.

6) Elute with 5 column volumes of elution buffer. Other volumes may be required if the interaction is difficult to break.

2.4 Analysis

Identify the fractions containing the protein. Use UV absorbance, SDS-PAGE, or western blot.

3. Cleaning-In-Place

In general, rProtein A/G Beads 4FF are well suited for reuse a number of times. When precipitation and protein aggregation cause the loss of velocity and combined loads, you need to clean the medium.

Remove the precipitation or denatured protein

Wash the column with 2 column volumes 6M guanidine hydrochloride solution. Finally wash the column with 5 column volumes 1XPBS (pH7.4).

Remove the non-specific adsorption protein

Wash the column with 3-4 column volumes 70% ethanol or 2 column volumes 0.1% non-ionic detergent. Finally wash the column with 5 column volumes 1XPBS (pH7.4).

4. Troubleshooting

5. Related Products