Immunoprecipitation with Dynabeads Protein G

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 (DynaMag-2 magnet) the generated Dynabeads Protein G-Ig complex is concentrated at the tube wall and the supernatant containing unwanted components can be discarded.

Table 1: Binding strength of protein G to Ig’s from different species. Please note that different monoclonals will   show different affinities towards protein G.
 
Following binding, the captured Ig’s can be eluted from the beads resulting in a pure and concentrated Ig sample for downstream applications such as antibody labeling or epitope mapping. Alternatively the Dynabeads Protein G-Ig complex can be used directly in immunoprecipitation of a target antigen/protein. The Dynabeads Protein G-Ig complex is added directly to the sample (cell lysate, acites, serum etc.) containing the target antigen and incubated for Dynabeads Protein G-Ig-antigen complex formation. The resulting Dynabeads Protein G-Ig-antigen complex can further be used in co-immunoprecipitation experiments, or the antigen can be directly eluted. Please note that during elution the affinity bound Ig’s will co-elute together with the target antigen. For several downstream applications such as SDS-PAGE followed by autoradiography or Western blotting the presence of Ig’s usually does not interfere with the experiment. However, if your downstream application involves purification of your target antigen, it might be an advantage to cross-link the Ig’s to protein G before immunoprecipitation in order to prevent co-elution of Ig’s (figure 1). Crosslinking is also required if the Dynabeads Protein G-Ig complex is to be re-used. The immunoprecipitation protocols are facilitated by the use of the magnet as described above.

Dynabeads Protein G are uniform, superparamagnetic polymer beads with protein G covalently coupled to the surface. The protein G employed is a recombinant streptococcal protein G lacking the albumin binding region. The molecular weight of recombinant protein G is 17 kD. Dynabeads diameter: 2.8 µm ± 0.2 µm (CV< 3%) Density: ~1.3 g/cm³ Surface area: 2-5 m²/g Dynabeads
 
Material supplied

Dynabeads Protein G are supplied in phosphate buffered saline (PBS), pH 7.4, containing 0.1%
Tween-20 and 0.02% sodium azide (NaN₃).

Cat. no. 10003D: 1 ml
Cat. no. 10004D: 5 ml
 
Additional Material Required

Magnet

For example, DynaMag-2 magnet (Cat. no. 12321D) for 20 µl to 2 ml samples

Mixer

Allowing tilting and rotation of tubes e.g. HulaMixer Sample Mixer (Cat. no. 15920D)
 
Buffers and Reagents

Washing Buffer
Citrate-Phosphate buffer, pH 5.0
 
Recipe for Washing buffer

Citrate-Phosphate buffer, pH 5.0:
4.7 g Citric Acid (MW=192)
9.2 g Dibasic Sodium Phosphate (Na₂HPO₄)dihydrate (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.
 
Elution Buffer

e.g. 0.1 M citrate (pH 2-3)
 
Cross-linking Buffers and Reagents

0.2 M triethanolamine, pH 8.2
20 mM DMP (freshly made)
50 mM Tris, pH 7.5
PBS/0.01-0.1 % Tween-20
 
Immunoprecipitation Buffer    

PBS
 
Aggregation of Dynabeads: To prevent aggregation of the beads with immobilized Ig or protein, 0.01-0.1% Tween-20 can be added to the storage buffer.
 
Re-use of Dynabeads Protein G: After elution of Ig’s Dynabeads Protein G can be reused at least five times. For re-use after elution, the Dynabeads Protein G should immediately be brought to neutral pH using a 0.1 M Na-phosphate buffer pH 8.0.

The amount of Ig captured is dependent on the concentration of Ig and Dynabeads Protein G in the starting sample. Dynabeads Protein G have successfully been used with a wide range of Ig concentrations in the coating reaction, ranging from 2 µg/ml to 2.5 mg/ml. It is recommended to keep the concentration of Dynabeads Protein G in the sample close to its original concentration in the Dynabeads Protein G vial. The binding efficiency will decrease if the Dynabeads Protein G are suspended in more than 5 times the original volume pipetted from the vial during the binding procedure. It is therefore recommended to keep the total reaction volume low in order to maintain as high concentration of Ig and Dynabeads Protein G as possible.

Maximum amount of Ig-binding is usually obtained after 40 min incubation. However, for some applications an incubation time of only 10 min might be sufficient. 

The procedure described below is for the isolation of Ig’s from a 100 µl sample containing between 0.2 µg (2 µg/ml) and 250 µg (2.5 mg/ml) Ig’s. The protocol can be scaled up and down as required.
 
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.

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.

The captured Ig’s are now ready to be eluted off the Dynabeads Protein G or the Dynabeads Protein G-Ig complex can be further used for immunoprecipitation.
 
Ig Elution Procedure

Elution of Ig’s is, in this example, performed by lowering pH using 0.1 M citrate (pH 2-3). The degree of acidity needed depends on the species and Ig subclass, but at pH 3.1 most Ig’s will be eluted off.

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).

Immunoprecipitation

Immunoprecipitation can be preformed by direct addition of the Dynabeads Protein G-Ig complex to a sample containing the target protein/antigen, or by first cross-linking the Ig’s covalently to protein G on the bead surface.
 
Cross-linking of Ig’s to Dynabeads Protein G

For some immunoprecipitation experiments coelution of Ig’s with the target antigen is not desired. To prevent co-elution, Ig’s can be cross-linked to Dynabeads Protein G. Cross-linking is also necessary if the Dynabeads Protein G-Ig complex is to be reused for immunoprecipitation.

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.

Trace amounts of Ig which are not successfully cross-linked can be eluted off prior to immunoprecipitation following the Ig elution procedure.
 
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.

Target Protein Elution Procedures

Most proteins/antigens will be eluted at pH 2.0-3.0 following the Ig elution procedure as described. However all conventional elution methods can be applied for the elution of target protein from the Dynabeads Protein G-Ig complex. Low pH, change in ionic strength, affinity elution etc. can be applied, or even boiling the beads in SDS-PAGE application buffer for direct characterization of protein on SDS-PAGE. The method of choice depends on the Ig’s affinity for the protein, stability of target protein and downstream applications and detection methods. If an elution method mild enough not to adversely denature the Ig is used, the Dynabeads Protein G-Ig complex can be reused.

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