实验概要

Protein G, a cell wall  component produced by group G Streptococcus strains, binds the Fc part  of a wide range of immunoglobulins (Ig’s). Protein G coupled to  superparamagnetic Dynabeads® is therefore suitable for easy and  efficient capture of a wide range of Ig’s. Protein G shows different  affinity for Ig’s from different species and for different isotypes  within a species (Table 1). An Ig containing sample is added to a tube  containing pre-washed Dynabeads® Protein G. During a short incubation  the Ig’s bind to Dynabeads® Protein G via their Fc part. By placing the  tube with the sample on a magnet (Dynal® MPC™) the generated Dynabeads®  Protein G-Ig complex is concentrated at the tube wall and the  supernatant containing unwanted components can be discarded. 

主要试剂

Dynal® MPC™ e.g. Dynal® MPC™-S

Washing Buffer

Citrate-Phosphate buffer, pH 5.0

Citrate-Phosphate buffer, pH 5.0:

4.7 g Citric Acid (MW=192)

9.2 g Dibasic Sodium Phosphate (Na₂HPO₄)dehydrate  (MW=178) Fill up to 1 litre with distilled water. In the protocol we  recommend to use Citrate Phosphate buffer pH 5.0 however it is also  possible to use other buffers like 0.1 M Na-citrate pH 5.0 or 0.1 M  Na-acetate pH 5.0.

 PBS

实验步骤

1.        Washing Procedure

1)        Resuspend the Dynabeads® Protein G thoroughly to obtain a homogeneous suspension.

2)         Transfer the desired volume of Dynabeads® Protein G to a tube at room  temperature. In order to isolate Ig from a 100 µl sample it is generally  recommended to use 20-100 µl of the Dynabeads® Protein G. A higher  volume can be used if the sample has a high Ig concentration or the Ig’s  are precious.

3)         Place the tube on the magnet for 1 min and discard the supernatant by  aspiration with a pipette while the tube remains on the magnet.

4)         Remove the tube from the magnet, add 0.5 ml of a Citrate-Phosphate  Buffer, pH 5.0 (see Material section for recipe) and resuspend the  Dynabeads® Protein G.

5)        Repeat steps 3, 4 and 3.

2.        Ig Capture Procedure

1)        Add 100 µl sample containing Ig’s to the washed Dynabeads® Protein G.

2)        Incubate with gentle mixing for 40 min at room temperature.

3)        Place the test tube on the magnet for 2 min and discard the supernatant.

4)         Remove the test tube from the magnet and add 0.5 ml Citrate-Phosphate  Buffer, pH 5.0. (For downstream immunoprecipitation or storage of  Dynabeads® Protein G, 0.01-0.1% Tween-20 can be added to the buffer to  prevent aggregation.)

5)        Wash the Dynabeads® Protein G by repeating steps 3 and 4 twice.

6)        Place the test tube on the magnet for 2 min and discard the supernatant.

3.        Ig Elution Procedure

1)        Add 30 µl 0.1 M citrate (pH 2-3) to the Dynabeads® Protein G-Ig complex.

2)        Mix well by tilting and rotation for 2 min.

3)        Place the test tube on the magnet for 1 min and transfer the supernatant containing purified Ig’s to a new tube.

4)         Repeat step 1, 2, and 3 in order to elute any remaining Ig. Pool the  supernatants containing the pure Ig’s (total collected volume = 60 µl).

4.        Cross-linking of Ig’s to Dynabeads® Protein G

1)        Wash the Dynabeads® Protein G-Ig complex twice in 1 ml 0.2 M triethanolamine, pH 8.2 with the use of a magnet.

2)         Resuspend the beads in 1 ml freshly made 20 mM DMP (dimethyl  pimelimidate x 2HCl) in 0.2 M triethanolamine, pH 8.2 (5.4 mg DMP/ml  buffer).

3)        Incubate with gentle mixing for 30 min at 20°C. Place the tube on the magnet for 1 min and discard the supernatant.

4)         Remove the tube from the magnet and stop the reaction by resuspending  the beads in 1 ml 50 mM Tris, pH 7.5, and incubate for 15 min with  gentle mixing.

5)        Place the tube on the magnet and discard the supernatant.

6)        Wash the now cross-linked beads 3 times with 1 ml PBS/0.01-0.1% Tween-20 with the use of a magnet.

7)         Resuspend the beads in 100 µl PBS/0.01-0.1 % Tween-20 or add the  protein containing sample directly to the cross-linked beads.

5.        Binding of Target Antigen to Dynabeads® Protein G-Ig complex

The  final yield will depend on the concentration of the target antigen, the  concentration of Dynabeads® Protein G-Ig complex, the affinity of the  immobilized Ig for the target protein/antigen and incubation time. For a  100 kD protein it is recommended to use a volume containing approximate  25 µg target protein/ml Dynabeads® Protein G (originally pipetted from  the vial) to assure an excess of protein. If dilution of protein is  necessary, PBS or 0.1 M phosphate buffer (pH 7.0) can be used as the  dilution buffer.

1)        Add the sample containing your target protein/ antigen to the Dynabeads®-Ig complex.

2)         Incubate the mixture with tilting and rotation for 1 hour at 2-8°C (for  concentrated samples an incubation time of 10 min might be sufficient.)

3)         Place the tube on the magnet for 2 min to collect the Dynabeads®  Protein G-Ig-antigen complex at the tube wall. For viscous samples,  double the separation time. Discard the supernatant.

4)        Wash the complex 3 times in 1 ml PBS with the use of a magnet.