Immunoprecipitation with Dynabeads Protein A

Immunomagnetic  protein  isolation  using  Dynabeads® Protein A provides a fast and reliable method for capturing Ig for small scale purification or downstream immunoprecipitation. Ig can be isolated directly from ascites, serum, tissue culture supernatants or other samples.

An Ig-containing sample is added to a tube containing pre-washed Dynabeads® Protein A. During a short incubation, the immunoglobulins will bind to Dynabeads® Protein A via their Fc part. Place the test tube on a magnet (Dynal® MPC™) to collect the Dynabeads® Protein A - Ig complex at the tube wall, and discard the supernatant.

The purified and concentrated Ig can be eluted off in a small volume for downstream use such as antibody labelling or epitope mapping. The Dynabeads® Protein A - Ig complex can also be used directly to immunoprecipitate a target antigen, or for immunodepletion (see References). Add the Dynabeads® Protein A - Ig complex directly to a sample (cell lysate or other) containing your target antigen and incubate for antibody-antigen  complex  formation.  Place  the  test tube on a magnet to collect the complex at the tube wall, and discard the supernatant. Resuspend the beads in a small volume for further use, or elute off your target protein directly e.g. in an acidic buffer or boil in a small volume of SDS-PAGE application buffer. If  your downstream application involves purification  of  your  target  protein,  you  might  want to cross-link the Ig to the protein A on the Dynabeads® before immunoprecipitation to prevent co-elution of the Ig. This is not necessary for downstream SDS-PAGE followed  by  autoradiography  or  Western blotting, and optional for Silver or Coomassie staining.

Dynabeads® Protein A are uniform, magnetizable polystyrene beads covalently coupled with recombinant
protein A. Typical characteristics for any given lot of this product:

• Diameter: 2.8 µm ± 0.2 µm (C.V. max 3%) Density: approx. 1.3 g/cm3
• Surface area: 3-9 m2/109 Dynabeads®
• The beads are supplied in phosphate buffered saline (PBS), pH 7.4, containing 0.1% Tween-20 and 0.02% sodium azide (NaN₃).

Protein A
Protein A has a high specificity for immunoglobulins (Table 1) and is therefore suitable for the one-step capture of Ig (1). The native bacterial cell wall protein is a single polypeptide chain of 42 kDa with four Ig Fc binding sites, two of which are active (2). The protein A employed in this product is a 45 kDa recombinant protein  containing  all  four  binding sites for the Fc region of Ig, but without any albumin binding sites.

Table 1: Binding strength of protein A to different species of immunoglobulins (Ig) and their subclasses. Monoclonal antibodies will vary in their affinity towards protein A.
 
Binding Capacity
Binding of Ig to protein A in solution is an equilibrium reaction. In order to capture as many Ig as possible, it is important to keep the reaction volume low to maintain high concentrations of beads and Ig. There is no need to pre-treat or dilute the sample (even viscous samples). For both the immobilization of Ig and downstream immunoprecipitation procedures, it is recommended to keep the concentration of beads in the sample close to its original concentration in the Dynabeads® Protein A vial. The binding efficiency will decrease if the Dynabeads® are suspended in more than 5 times the original volume of Dynabeads® initially added. The amount of Ig captured is dependent on the concentration of Ig in the starting sample. 100 µl Dynabeads® Protein A will isolate approximately 25-30 µg human IgG from a sample containing 20-200 µg IgG/ml. A higher volume of Dynabeads® is recommended to avoid waste of Ig when working with concentrated samples or the Ig is precious. Keep all other parameters fixed as described below. Maximum amount of Ig-binding is obtained after 10 minutes

Dynabeads® Protein A should be washed prior to use. Washed Dynabeads® Protein A are resuspended in a basic phosphate buffer (e.g. pH 8.1) to facilitate binding of Ig to beads. The pH in the sample containing Ig might be adjusted for the same reason using a basic 5 x stock solution. The procedure described below is for the isolation of approximately 25 µg IgG from 10 µl human serum (or similar sample). The use of polypropylene tubes is recommended for washing and Ig capture.

Washing Procedure
The washing procedure is facilitated by the use of a magnet (Dynal® MPC).

1. Resuspend the Dynabeads®  Protein A, thoroughly in the vial (e.g. by vortexing 1-2 minutes or rotating on a roller) to obtain a homogeneous suspension.
2. Transfer 100 µl Dynabeads® Protein A to a test tube at room temperature. (Please refer to Materials section for details on binding capacity.)
3. Place the test tube on the magnet for one minute and pipette off the supernatant.
4. Remove the test tube from the magnet and add 0.5 ml 0.1 M Na-phosphate buffer pH 8.
5. Repeat steps 3, 4 and 3.

Ig Capture Procedure
In this example, the Dynabeads® volume is much larger than the sample volume. In cases where the sample volume is as large as 1/4 the Dynabeads® volume, add 0.5 M Na-phosphate 5 x stock solution to raise the pH in the sample to 8 (with a final molarity of 0.1) before adding to the Dynabeads® (i.e. ad 10 µl stock solution to each 40 µl sample). Dynabeads® are resuspended in an adjusted volume so that sample and Dynabeads® volumes together is the same as the bead-volume originally pipetted from the vial.

1. Resuspend the washed Dynabeads® in 90 µl 0.1 M Na-phosphate buffer pH 8.
2. Add 10  µl  serum to the solution  containing Dynabeads®.
3. Incubate with slow tilt rotation mixing for 10 minutes at room temperature.
4. Place the test tube on the magnet for 2 minutes and pipette off the supernatant.
5. Remove the test tube from the magnet and add 0.5 ml 0.1 M Na-phosphate buffer pH 8. (For downstream immunoprecipitation or storage of Dynabeads®, 0.01-0.1% Tween-20 can be added to  the  buffer  to  prevent  aggregation  of  the Dynabeads® Protein A - Ig complex.)
6. Repeat steps 4, 5, 4, 5, 4.The purified Ig is now ready to be eluted off the Dynabeads® (see Ig Elution Procedure below)  or  the  Dynabeads® Protein A - Ig complex can be used for immunoprecipitation - either by adding directly to a new sample containing the target protein, or by first cross-linking the Ig covalently to the protein A on the Dynabeads® (see Immunoprecipitation below).

Ig Elution Procedure
Eluting Ig off the Dynabeads® Protein A 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 depends on the species and Ig sub-class, but at pH 3 most Ig will be eluted.

1. Add an appropriate amount (e.g. 40 µl) 0.1 M citrate (pH 2-3) to the Dynabeads® Protein A - Ig complex with immobilized IgG.
2. Mix well by tilting and rotation 2 minutes.
3. Place the test tube on a magnet and transfer the supernatant, containing purified Ig, to a clean tube. Immediately adjust the eluate to physiologic pH by adding alkaline buffer (e.g. 1M Tris pH 7.5-9).

Immunoprecipitation
When isolating antigens for SDS-PAGE followed by Western blotting or autoradiography the presence of Ig will not disturb your detection system. For other applications (e.g. protein purification, amino acid sequencing or when the Dynabeads® Protein A with bound Ig is to be reused) co-elution of the Ig is not desired. To prevent this, the captured Ig can be crosslinked to the protein A on the Dynabeads®. Cross-linking is also necessary if the Dynabeads® - Ig complex is reused for immunoprecipitation.

Crosslinking
The protocol presented below is an example using one of several commercially available cross-linkers.

1. Add 1 ml 0.2 M triethanolamine, pH 8.2 to the Dynabeads® — Ig complex with immobilized immunoglobulin. Wash twice according to procedure 2.1 above, using 0.2 M triethanolamine, pH 8.2 as the washing buffer.
2. Resuspend the Dynabeads® - Ig complex 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 Dynabeads® - Ig complex.
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 Dynabeads®  - Ig complex 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® - Ig complex 3 times with 1 ml PBS pH 7.4 by the use of a magnet, according to procedure 2.1 above. Resuspend the Dynabeads® - Ig complex to 100 µl or add directly to antigen-containing solution. The full recovery of your Ig activity cannot be guaranteed, as this varies from Ig to Ig.
7. Note: 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

Binding of Antigen
Trace amounts of Ig not cross-linked to Dynabeads® Protein A can be removed prior to binding by following the elution procedure described in 2.3 above. Binding of protein or other antigen to the Dynabeads® - Ig complex is dependent on the concentration of the Dynabeads®, antigen concentration, the affinity of the immobilized Ig and incubation time. Binding is performed at 2-8°C from 10 minutes to 1 hour. Equilibrium antibody-antigen is reached at approximately 1 hour.
 

1. Add   sample   containing   antigen   to   the Dynabeads® - Ig complex. For a 100 kD protein, use a volume containing approximate 25 µg target antigen per ml of beads to assure an excess of antigen. If dilution of antigen is necessary, PBS or 0.1 M phosphate buffer (pH 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 vial).
3. Place the tube on the magnet for 2 minutes to collect the Dynabeads® - Ig complex at the tube wall. For viscous samples, double the time on the magnet. Pipette off the supernatant.
4. Wash the Dynabeads® - Ig complex 3 times using 1 ml PBS each time and change buffers by the use of a magnet, according to procedure 2.1 above.

 
Target Protein Elution Procedure
Conventional elution methods can be applied for the elution of target antigen from the Dynabeads® -Ig complex. Low pH (2-3), change in ionic strength, affinity elution, electrophoresis, polarity reducing agents, deforming eluents 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 antigen, stability of target protein and downstream applications and detection methods. Most antigens will be eluted at pH 3 following the procedure described under 2.3 above. If the Dynabeads® - Ig complex is to be reused, mild elution methods should be employed. To prevent aggregation of the beads with immobilized Ig, 0.01-0.1% Tween-20 can be added to the storage buffer.
 
Re-use of Dynabeads® Protein A
For re-use after elution, the Dynabeads® Protein A should be brought to neutral pH using a Na-phosphate buffer, pH 7.
 
Additional Material Required

• Magnet: Dynal® MPC™ e.g. Dynal® MPC™-S (Cat. no. 120.20D) for 20 µl to 2 ml samples
• Mixer: Allowing tilting and rotation of tubes e.g. Dynal® MX1 (Cat. no. 159.07) or Dynal® Sample Mixer (Cat. no. 947.01)
• Buffers and reagents

1. Tejeda-Mansir A et al. Bioprocess Engineering 1997;17: 39-44

Additional references where Dynabeads Protein A have been used:


2. Lin-Lee Y-C et al. Nuclear localization in the biology of the CD40 receptor in normal and neoplastic human B lymphocytes. J. Biol. Chem. 2006;281: 18878-18887
3. Yuan X et al. Androgen receptor remains critical for cell cycle progression in androgen-independent CWR22 prostate cancer   cells. Am. J. Pathol. 2006;169:682-696
4. Haren L et al. NEDD1-dependent recruitment of the gamma-tubulin ring complex to the centro-some is necessary for centriole duplication and spindle assembly. J. Cell Biol. 2006;172:505-515
5. Kudva IT et al. Identification of a protein subset of the Anthrax spore immunome in humans immunized with the Anthrax vaccine adsorbed preparation. Infect. Immun. 2005;73:5685-5696
6. Pham LV et al. Constitutive NF-kappaB and NFAT activation in aggressive B-cell lymphomas synergistically activates the CD154 gene and maintains lymphoma cell survival. Blood 2005;106(12):3940-3947
7. Wu Z et al. Interleukin-21 receptor gene induction in human T cells Is mediated by T-cell receptor-induced Sp1 activity. Mol. Cell. Biol. 2005;25:9741-9752
8. Wan L et al. The survival of motor neurons protein determines the capacity for snRNP assembly: Biochemical deficiency in spinal muscular atrophy. Mol. Cell. Biol. 2005;25(13):5543-5551
9. Feng W et al. Gemins modulate the expression and activity of the SMN complex. Hum. Mol. Genet. 2005;14:1605-1611
10. Catrein I et al. Experimental proof for a signal peptidase I like activity in Mycoplasma pneumoniae, but absence of a gene encoding a conserved bacterial type I SPase. FEBS J. 2005;272:2892-2900
11. Losada A et al. Functional contribution of Pds5 to cohesin mediated cohesion in human cells and Xenopus egg extracts. J. Cell Sci. 2005;118:2133-2141
12. Kops GJP et al. ZW10 links mitotic checkpoint signalling to the structural kinetochore. J.Cell Biol. 2005;169:49-60
13. Maehara K et al. Reduction of total E2F/DP activity induces senescence-like cell cycle arrest in cancer cells lacking functional pRB and p53. J.Cell Biol. 2005;168:553-560
14. Akbari M et al. Repair of U/G and U/A in DNA by UNG2 associated repair complexes takes place predominantly by short-patch repair both in proliferating and growth-arrested cells. Nucl. Ac. Res. 2004;32:5486-5498
15. Sáez-Vasquez J et al. A plant snoRNP complex containing snoRNAs, fibrillarin, and nucleolin-like proteins is competent for both rRNA gene binding and pre-rRNA processing in vitro. Mol. Cell. Biol. 2004;24:7284-7297
16. Fukui T et al. Distinct roles of DNA polymerases delta and epsilon at the replication fork in Xenopus egg extracts. Genes Cells 2004;9:179-191
17. Lin C-W and Engelman A. The barrier-to-autointegration factor is a component of functional human immunodeficiency virus type 1 preintegration complexes. J. Virol. 2003;77:5030-5036
18. Huang C et al. Parallel activation of phosphatidylinositol 4-kinase and phospholipase C by the extracellular   calcium-sensing receptor. J. Biol. Chem. 2002;277:20293-20300
19. Rajan S et al. Interaction with 14-3-3 proteins promotes functional expression of the potassium channels TASK-1 and TASK-3. J.Physiol. 2002;545,13-26
20. Koizume S et al. Heterogeneity in the modification and involvement of chromatin components of the CpG island of the silenced human CDH1 gene in cancer cells. Nucl. Ac. Res. 2002;30:4770-4780
21. Huang C et al. Parallel activation of phosphatidylinositol 4-kinase and phospholipase C by the extracellular   calcium-sensing receptor. J. Biol. Chem. 2002;277:20293-20300
22. Popov AV et al. XMAP215 regulates microtubule dynamics through two distinct domains. EMBO J. 2001;20:397-410
23. Budde PP et al. Regulation of Op18 during spindle assembly in Xenopus egg extracts. J. Cell  Biol. 2001;153(1):149-158