M-280 Sheep anti-Rabbit IgG - binding immunoglobulins (Ig) - target molecules

• Magnetic device (Dynal® MPC, Magnetic Particle Concentrator).
• Mixing/rotation device
• Vortex mixer
• Primary antibody
• Test tubes and pipettes
• Buffers/solutions

Washing Procedure

The Dynabeads® M-280 Sheep anti-Rabbit IgG should be washed before use to remove preservatives. The washing procedure is facilitated by the use of a magnet. Make sure the beads are thoroughly resuspended to a homogeneous solution before use (in step 2 below).

1. Resuspend the Dynabeads® M-280 Sheep anti-Rabbit thoroughly in the vial by vortexing and/or shaking.


2. Transfer the required amount of resuspended beads into a tube.
   


3. Place the tube on the magnet for 2 minutes and pipette off the supernatant. Avoid touching the inside wall of the tube (where the beads attract to the magnet) with the pipette tip.


4. Remove the tube from the magnet, and resuspend the pellet in an excess volume of washing buffer (see below).


5. Repeat step 3 and again resuspend the washed beads in washing buffer. The washed Dynabeads® M-280 Sheep anti-Rabbit IgG can be resuspended in any chosen volume. A small volume should be chosen (e.g. the volume originally pipetted from the vial) so that a final bead concentration of 1-2 x 10⁷ can be achieved for efficient target isolation.

Separation of Target Ig

1. Resuspend the washed Dynabeads® M-280 Sheep anti-Rabbit IgG well by vortexing or shaking.


2. Add the sample containing your target-Ig to the washed beads. Approx. 0.1-1 μg Ig/10⁷ beads may be sufficient for binding.


3. Incubate with slow tilt rotation mixing for 30 min or up to 24 hours at 2-8°C.


4. Place the test tube on the magnet for 2 minutes and pipette off the supermantant.


5. Remove the test tube from the magnet, add washing buffer and resuspend.


6. Repeat steps 4 and 5 twice, place the tube on the magnet and remove the supernatant.


7. The bound and purified Ig is now ready to be eluted off the beads (see below), or the Ig-coated beads can be used for immunoprecipitation (see below). The latter is done by either adding the coated beads directly to a new sample containing the target protein, or by first covalently crosslinking the primary Ig to the sheep anti-rabbit IgG on the beads.

Elution of Isolated Ig

1. Eluting the isolated Ig off the Dynabeads® M-280 Sheep anti-Rabbit IgG is - in this example - performed by lowering pH using 0.1 M citrate (pH 2-3) as the elution buffer. The degree of acidity needed will depend on the specific Ig, but at pH 3.1 most Ig will be eluted off.


2. Add 0.1 M citrate (pH 2-3) to Dynabeads® M-280 Sheep anti-Rabbit IgG with immobilised Ig.


3. Mix well by tilting and rotation for 2 minutes.


4. Place the test tube on a magnet and transfer the supernatant, containing purified Ig, to a clean tube.


5. Again add 0.1 M citrate (pH 2-3) to the beads to elute any remaining Ig.


6. Mix well by tilting and rotation for 2 minutes.


7. Place the test tube on a magnet, pipette off the eluate and pool the supernatants containing pure Ig.


8.  Dynabeads® M-280 Sheep anti-Rabbit IgG where the Ig has been eluted off can be reused at least five times. For re-use after elution, the beads should immediately be brought to neutral pH using a Na-phosphate buffer. For storage, the beads should be resuspended in a PBS/BSA buffer (see section below).

Immunoprecipitation

Bound Ig will be co-eluted along with the target using different elution methods (see below). When isolating antigens, for SDS-PAGE followed by Western blotting or autoradiography, the presence of Ig may not disturb the detection system. For other applications such as protein purification or amino acid sequencing, co-elution of the I  is not desired. If the presence of Ig will not disturb your detection system, go directly to section below. For applications where co-elution of the Ig is not desired, your primary Ig can be crosslinked to the sheep anti-rabbit IgG as described in section below. If trace amounts of Ig is not crosslinked, this can be removed prior to immunoprecipitation by following the procedure described in section above. After isolation and elution of the target protein, the Ig-coated beads may be reused for immunoprecipitation. If the Ig-coated beads are to be reused, crosslinking is necessary.

Crosslinking Ig to the Beads

The protocol presented here uses 0.2 M triethanolamine pH 8.2. Other non-amine containing buffers with pH 7-9 can also be used.

1. Add 1 ml 0.2 M triethanolamine, pH 8.2 to the Dynabeads® M-280 Sheep anti-Rabbit IgG with immobilised Ig. Wash twice using a magnet and 0.2 M triethanolamine, pH 8.2 as the washing buffer (see section above).


2. Resuspend the beads in 1 ml of 20 mM DMP (dimethyl pimelimidate dihydrochloride, Pierce #21666) in 0.2 M triethanolamine, pH 8.2 (5.4 mg DMP/ml buffer). This crosslinking solution must be prepared immediately before adding to the beads.


3. Incubate with rotational mixing for 30 minutes at 20°C. Place the tube on the magnet and discard the supernatant.


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


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


6. Wash the now crosslinked Dynabeads® 3 times with 1 ml PBS/BSA by the use of a magnet (see section above). Pre-elution is optional.  Resuspend the beads in chosen volume or add directly to antigen-containing solution. Invitrogen Dynal® can not guarantee the full recovery of your Ig activity, as this varies from Ig to Ig.

Antigen-Binding to Ig-Coated Beads

Binding of protein or other antigen to primary Ig-coated Dynabeads® is dependent on the concentration of the beads, antigen concentration, the affinity of the immobilised Ig and time. Binding is performed at 2-8°C from 10 minutes to 1 hour. Equilibrium antibody-antigen is reached after approximately 1 hour.

1. Add sample containing antigen to the beads. For a 100 kD protein, use a volume containing approximate 25 μg target antigen/ml beads to assure an excess of antigen. If dilution of antigen is necessary, PBS or 0.1 M phosphate buffer (pH 7-8) can be used as dilution buffer.


2. Incubate with tilting and rotation for one hour. (Incubation times as low as 10 minutes can be used with concentrated protein samples in volumes close to what was originally pipetted from the product vial).


3. Place the tube on the magnet for 2 minutes to collect the Ig-coated Dynabeads®-target complex at the tube wall. For viscous samples, double the time on the magnet. Pipette off the supernatant.


4. Wash the beads 3 times using 1 ml PBS each time and change buffers by the use of a magnet (see section above).

Target Protein Elution

Conventional elution methods can be applied for the elution of target antigen from the Dynabeads®, and one of the major advantages using Dynabeads® in protein/Ig isolation is the possibility to elute in small volumes. Low pH (2.8–3.5), change in ionic strength, affinity elution, electrophoresis, polarity reducing agents, deforming eluents can be applied, or even boiling the bead-target complex in SDS-PAGE application buffer for direct characterisation of protein on SDS-PAGE.

The method of choice depends on the Ig’s affinity for the specific target protein, stability of target protein and downstream applications and detection methods. Most proteins will be eluted at pH 3.1 following the procedure described under Dynabeads® Ig elution procedure (see section above). Some protein functionality might be lost under these conditions. If maintaining functionality of the target protein is important, try milder elution conditions first such as high salt (e.g. 2M NaCI) or step-wise elution reducing pH from 6 down to 3. This is also recommended if the bead-bound ligand must remain functional to allow reuse of the Dynabeads®.

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