rProtein AG Beads 4FF

rProtein A/G Beads 4FF                

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.

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 or 0.45 μm filter before use.

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

Acid Elution Buffer: 0.1 M glycine, pH 3.0

Neutralization Buffer: 1 M Tris-HCl, pH 8.5

Coupling Buffer: 0.2 Mtriethanolamine, pH 8.2

Cross-linking agent: DMP（dimethyl pimelimidate dihydrochloride）

Stop Buffer: 50 mM Tris, pH 7.5

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 with Binding/Wash Buffer.

For adherent cells in a 100 mm culture dish, the cell culture medium was removed and washed once with pre-cooled PBS, then 2ml cell lysis buffer was added to lyse the cells. For suspension cells, the cells were collected by centrifugation, and then washed with PBS once for lysis refer to adherent cells. Plant or animal tissue samples can be decomposed by liquid nitrogen grinding. Please refer to the instructions of different lysates for the specific lysis method. The final total protein concentration of the lysate is appropriate in the range of 0.5-1 ug/ul. In general, the expression of target protein is different, so the total protein concentration needs to be adjusted by pre-experiment.

2.3 Remove Nonspecific Binding (Optional)

1) Take 200 μl to 1 ml of protein samples. Add 1 mg Normal IgG with the same species of IgG used in immunoprecipitation and 20 μl of fully re-suspended rProtein A/G Beads 4FF. The samples were shaken slowly for 30 minutes at 4℃.

2) Centrifuge at 2500 rpm (about 1000 g) for 1 minute, and take the supernatant for immunoprecipitation.

Note: There are several components in mammalian cells that bind to IgG, and nonspecific bands may appear in subsequent western blots. Pretreatment of lysate using Normal IgG and rProtein A/G Beads 4FF may reduce nonspecific adsorption.

2.4 Immuneprecipitation

2.4.1 Antibody adsorption

1) Completely resuspend rProtein A/G Beads 4FF and add 100 μl to 1.5-2 ml micro centrifuge tube. Centrifuge for 1 minute at 800 rpm and discard supernatant.

2) Add 0.5 ml of Binding Buffer, centrifuge for 1 minute at 800 rpm and discard supernatant. Repeat this step two times.

3) Add the sample containing target IgG to the tube and gently invert the tube to mix. Incubate the tube at room temperature with mixing (on a shaker or rotator) for 30minutes.

4) Centrifuge for 1 minute at 800 rpm and discard supernatant. Repeat this step two times. If necessary, keep the supernatant for analysis.

5) Add 500 μl Binding/Wash Buffer to the tube and mix well, Centrifuge for 1 minute at 800rpm and discard supernatant. Repeat this step two times.

2.4.2 Cross‐linking IgG to the Beads (Optional)

If you need to elute antibodies and target antigen together, please ignore this step.

• Add 1 ml 0.2 M triethanolamine, pH 8.2 to the rProtein A/G Beads 4FFwith immobilised IgG. Centrifuge for 1 minute at 800rpm and discard supernatant.
• Resuspend the beads in 1 ml of 20 mM dimethyl pimelimidate dihydrochloride (DMP) in 0.2 M triethanolamine, pH 8.2 (5.4 mg DMP/ml buffer). This cross‐linking solution must be prepared freshly.
• Incubate the tube at room temperature with mixing (on a shaker or rotator) for 30minutes. Centrifuge for 1 minute at 800 rpm and discard supernatant.
• Resuspend the beads in 1 ml of 50 mM Tris, pH 7.5 to stop the reaction and incubate for 15 minutes at room temperature with rotational mixing.
• Centrifuge for 1 minute at 800 rpm and discard supernatant.
• Wash the cross‐linked beads (Beads-Ab complex) three times with 500ul Wash Buffer.

2.4.3 Binding Antigen to the Beads-Ab complex

1) Add sample containing the antigen (Ag) (typically 100–1000 μl) and gently mix to resuspend the Beads-Ab complex.

2) Incubate with rotation for 30min at room temperature to allow Ag to bind to the Beads-Ab complex.

3) Centrifuge for 1 minute at 800 rpm and discard supernatant.Wash the beads three times with 1 ml PBS, pH7.4.

Note: Depending on the affinity of the antibody, it may be necessary to increase incubation times for optimal binding.

2.4.4 Elution of Target Protein

1. Denaturing elution

1) Add 50 μl 1XSDS-PAGE Sample Buffer to the tube and mix well.

2) Heat the tube at 100℃ for five minutes.

3) Centrifuge for 1 minute at 800 rpm and transfer the supernatant containing desired sample into a new tube.

4) Analyze the sample by SDS‐PAGE followed by Western blot analysis.

1. Non-denaturing elution

1) Add 150 μl Elution Buffer to the tube and mix well. Incubate for five minutes at room temperature with occasional mixing.

2) Centrifuge for 1 minute at 800 rpm and transfer the supernatant into a new tube.

3) Repeat Step 1 and 2 twice.

4) Add 10μl Neutralization Buffer to each 100 μl of eluate to neutralize the pH.

2.5 Co- Immunoprecipitation

• Mix antibody with the target protein, incubation for 30-60 min at room temperature or at 2-8 degree overnight .

Note: Depending on the affinity of the antibody, it may be necessary to increase incubation times for optimal binding.

• Completely resuspend rProtein A/G Beads 4FF and add 100μl to 1.5-2ml microcentrifuge tube. Centrifuge for 1 minute at 800 rpm and discard supernatant.

Add 0.5ml of Binding Buffer, centrifuge for 1 minute at 800 rpm and discard supernatant. Repeat this step two times.

• Mix the antibody-antigen with the beads. Incubate at room temperature with mixing (on a shaker or rotator) for 30minutes. Centrifuge for 1 minute at 800 rpm and collect supernatant for detection.
• Add 500 μl Binding/Wash Buffer to the tube and mix well, Centrifuge for 1 minute at 800 rpm and discard supernatant. Repeat this step two times.
• Elute the sample as 2.4.4.

3. Related Products