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Immunoprecipitation using Dynabeads®

Straightforward immunoprecipitation with Dynabeads® and a denaturing elution.

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Immunoprecipitation (IP) is a cornerstone technique for protein interaction and function studies, offering a window into the molecular dynamics within cells. This protocol guides you through the seamless integration of Dynabeads® technology for precise antibody-antigen complex isolation. From initial bead preparation to the final elution step, this method gives you a simple and reliable approach to dissecting protein interactions, ensuring high-quality, reproducible results. 

Materials

  • DynaMag™-2 Magnet (Invitrogen: 12321D)
  • Dynabeads® Protein A (Invitrogen: 10002D)
  • Washing Buffer: phosphate buffered saline (PBS) (pH 7.4) (Sartorius: 02-023-1A) with 0.02% Tween®-20 (Sigma: P1379) 
  • Sample Buffer (your choice, but we recommend Laemmli Buffer)
    • Note: fresh β-mercaptoethanol (βME) should be added to the Sample Buffer if this is your reducing agent of choice 
Laemmli Buffer x2 (50 ml)
Reagent Volume/weight % of final volume
Tris-Base (0.5 M, PH=6.8) 12.5 mL 25%
SDS 10 mL from 20%SDS 4%
Glycerol 10 mL 20%
2-Mercaptoethanol 5 µL 0.01%
Bromophenol Blue sodium salt 2.5 mg 0.16%
Double distilled water 17.49 mL 50%

Dynabeads® preparation 

  1. Resuspend Dynabeads®  by vortexing for 30 seconds. 
  2. Transfer 50 µL (1.5 mg) of Dynabeads® to an Eppendorf tube. 
  3. Place the Eppendorf tube on the DynaMag™-2 Magnet and remove the supernatant (Dynabeads® will bind to the wall of your Eppendorf tube). 
  4. Remove the Eppendorf tube from the magnet and proceed directly to “Antibody binding to Dynabeads”.

Antibody binding to Dynabeads®

  1. In parallel, add the appropriate amount of your antibody (Ab) (1–10 µg) and the same amount of the IgG isotype control – depending on protein abundance and Ab affinity – diluted in 200 µL Washing Buffer to the Dynabeads® prepared from the “Dynabeads® preparation” step. 
  2. Incubate with rotation for 10–30 minutes at room temperature. 
  3. Place the Eppendorf tube on DynaMag™-2 Magnet and remove the supernatant. 
  4. Separate the Eppendorf tube from the DynaMag™-2 Magnet and gently wash the Dynabeads®-Ab complex with 200 µL Washing Buffer. 
  5. Place the Eppendorf tube on DynaMag™-2 Magnet and remove the supernatant. 
  6. Proceed to “Immunoprecipitation”.  

Immunoprecipitation 

  1. Gently resuspend the Dynabeads®-Ab complex by adding your sample lysate containing your antigen (Ag) (1–4 mg in 100–1,000 µL) and in parallel with the tube of the IgG isotype control. 
  2. Incubate with rotation for 10–60 min – depending on protein abundance and Ab affinity – at room temperature to allow the binding of the target protein to the Dynabeads®-Ab complex. 
  3. Place the Eppendorf tube on DynaMag™-2 Magnet and remove the supernatant. 
  4. Gently resuspend the Dynabeads®-Ab-Ag complex in 200 µL of Washing Buffer. 
  5. Wash 3 times and separate the Eppendorf tube from DynaMag™-2 Magnet between the washes.  
  6. Resuspend the Dynabeads®-Ab-Ag complex in 100 µL of Washing Buffer and transfer to a new Eppendorf tube, to prevent elution of proteins bound to the Eppendorf tube wall. 
  7. Place the Eppendorf tube on DynaMag™-2 Magnet and remove the supernatant. 
  8. Proceed to “Denaturing elution”. 

Denaturing elution

  1. Separate the Eppendorf tube from DynaMag™-2 Magnet. Add 40 µL of Sample Buffer (e.g. Laemmli Buffer)(1X), gently pipette to resuspend the Dynabeads®-Ab-Ag complex. 
  2. Heat for 5 min at 100ºC. 
  3. Separate the Eppendorf tube from the DynaMag™-2 Magnet and load the supernatant onto a gel. 

Proceed to the Western Blot Protocol 

Example Data

Immunoprecipitation of rat brain lysates using SV2A and isotype control antibodies

  1. Brain lysate (input).
  2. Brain lysate + Protein A beads + Rabbit IgG Isotype Control (#RIC-001), (4µg).
  3. Brain lysate + Protein A beads + Anti-SV2A (#ANR-095), (4µg). 

Black arrow indicates the SV2A protein. Red arrow indicates the IgG heavy chain. Western blot was conducted using Anti-SV2A (#ANR-095), (1:500).