實驗概要
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.
主要試劑
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
實驗步驟
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.