Immunoprecipitation using Protein A - 10001D

This product features New & Improved Dynabeads Protein A. The new Dynabeads have optimized bead surface characteristics and a new buffer formulation to give better consistency, lower non-specific binding and improved handling properties. More details about the changes can be found on the following web page: www.thermofisher.com/immunoprecipitation

Intended Use

Dynabeads Protein A are designed for immunoprecipitation of proteins, protein complexes, protein-nucleic acid complexes and other antigens.

Principle

Antibody (Ab) is added to the Dynabeads Protein A. During a short incubation the Ab will bind to the Dynabeads® via their Fc region. The tube is then placed on a Dynal® magnet, where the beads will migrate to the side of the tube facing the magnet and allow for easy removal of the supernatant. The bead-bound Ab may now be used for immunoprecipitation. Bound material is easily collected utilizing the unique magnetic properties of the Dynabeads. Magnetic separation facilitates washing, buffer changes and elution

Description of Materials

Material supplied

Dynabeads Protein A are uniform, 2.8 μm, superparamagnetic beads with recombinant Protein A (approx. 45 kDa covalently coupled to the surface. The beads are supplied at a concentration of 30 mg/ml in phosphate buffered saline (PBS), pH 7.4, containing 0.01% Tween®-20 and 0.09% sodium azide (NaN₃).
Cat.no. 100.01D: 1 ml
Cat.no. 100.02D: 5 ml

Additional Materials Required

• Magnet: e.g DynaMag-2 (see www.thermofisher.com/magnets ) for further information and magnet recommendations)
• Mixer allowing tilting and rotation of tubes

The below are general recommendations, alternative buffers of your choice may also be used. Please see Technical Advice for details.

• Cell lysis buffer, e.g. Cell Extraction Buffer (Cat. no. FNN0011) or NP40 Cell Lysis Buffer (Cat. no. FNN0021) from Invitrogen
• PBS pH7.4
• PBS pH7.4 w/ 0.02% Tween 20
• 50 mM Glycine pH 2.8 (elution buffer)
• NuPAGE LDS Sample Buffer and NuPAGE Sample Reducing Agent (elution buffer)
  

Antibody selection

The choice of primary antibody is the most important factor for successful target antigen capture. Note that some antibodies may show reduced antigen-binding efficiency for immunoprecipitation, even though the antibody shows good results in other immunological assays. Please refer to the manufacturer’s recommendations regarding your primary antibody.

This protocol offers a general guideline for immunoprecipitation. Optimization may be required for each antibody and target antigen. The protocol uses 50 μl of Dynabeads® Protein A, but this may be scaled up or down as required.

Cell lysis

Cells may be lysed using any standard cell lysis protocol compatible with your starting material. We recommend the use of Cell Extraction Buffer or NP40 Cell Lysis Buffer. For protocols and additional information about cell lysis, please see www.lifetechnologies.com/immunoprecipitation

Preparation of Dynabeads®

1. Completely resuspend Dynabeads® by pipetting or by rotating on a roller (5 minutes).


2. Transfer 50 µl (1.5 mg) Dynabeads® to a tube.


3. Place the tube on the magnet to separate the beads from the solution and remove the supernatant.


4. Remove the tube from the magnet.


5. Proceed directly to Binding of Antibody.

Binding of Antibody
 

6. Add your antibody (Ab) (typically 1-10 µg) diluted in 200 µl PBS w/ Tween 20, to the tube from step 4 above. The optimal amount of Ab needed will depend upon the individual Ab used.


7. Incubate with rotation for 10 minutes at room temperature.


8. Place the tube on the magnet and remove the supernatant.


9. Remove the tube from the magnet and resuspend the beads-Ab complex in 200 µl PBS w/Tween 20. Wash by gentle pipetting.


10. Proceed to Immunoprecipitation
    
    Crosslinking
    
    If you wish to avoid co-elution of your antibody, you should crosslink your antibody to the Dynabeads® before continuing with immunoprecipitation. We recommend using the crosslinking reagent BS³. For further information and procedure, please visit: www.lifetechnologies.com/crosslinking
    
    Immunoprecipitation of Target Antigen


11. Place the tube (from step 9) on the magnet, and remove the supernatant.


12. Add your sample containing the antigen (Ag) (typically 100-1,000 µl) and gently pipette to resuspend the Dynabeads®-Ab complex.


13. Incubate with rotation for 10 minutes at room temperature to allow Ag to bind to the Dynabeads®-Ab complex. Note: Depending on the affinity of the antibody, it may be necessary to increase incubation times for optimal binding. Please see section "Technical Advice", for details.


14. Place the tube on the magnet. Transfer the supernatant to a clean tube for further analysis if desired.


15. Wash the Dynabeads®-Ab-Ag complex three times using 200 µl PBS for each wash. Separate on the magnet between each wash, remove supernatant and resuspend by gentle pipetting.


16. Resuspend the Dynabeads®-Ab-Ag complex in 100 µl PBS and transfer the bead suspension to a clean tube. This is recommended to avoid co-elution of proteins bound to the tube wall.


17. Proceed to Elution of Target Antigen
    
    Elution of Target Antigern
    
    Alternative protocols A or B:
    
    A. Denturing elution
    


18. Place the tube (from step 16) on the magnet and remove the supernatant.


19. Add 20 µl premixed NuPAGE LDS Sample Buffer and NuPAGE Sample Reducing Agent (mixed as per manufacturer's instructions). Gently pipette to resuspend the Dynabeads®-Ab-Ag complex. Heat for 10 min at 70°


20. Place the tube on the magnet and load the supernatant/sample onto the gel. Note: An alternative sample buffer of your choice may also be used (e.g. SDS sample buffer). Follow the manufacturer's recommended temperatures and heating times for these buffers prior to gel loading.
    
    B. Gentle, non-denaturing elution
    


21. Place the tube (from Step 16) on the magnet and remove the supernatant.


22. Add 20 µl elution buffer and gently pipette to resuspend the Dynabeads®-Ab-Ag complex. Avoid foaming.


23. Incubate with rotation for 2 minutes at room temperature to dissociate the complex.


24. Place the tube on the magnet and transfer the supernatant containing elution to a clean tube. If the eluted protein is to be used for functional assays or stored , the pH of the eluate can be adjusted by adding 1M Tris, pH 7.5.

Binding characteristics:

Binding capacity:

Dynabeads® Protein A have a binding capacity of approximately 8 μg human IgG per mg beads. The amount of Ab captured depends on the concentration of Ab and Dynabeads® Protein A in the starting sample.

Table 1. Binding strength of Protein A and G to different species of Igs and their subclasses

Buffer recommendations

For standard IP we recommend using PBS for antibody binding and washing steps. However, these may be substituted by other buffers of choice, such as alternative phosphate buffers, lysis buffer (e.g. RIPA, NP40), HEPES, Tris etc. The recommended elution buffer may also be substituted by alternative low pH-, high pH- or high salt buffers, depending on your target protein and downstream application.

Low affinity antibodies

When working with low-affinity antibodies which require increased incubation times, it can be preferable to pre-incubate sample and antibody prior to bead capture (Indirect Approach). This improves binding kinetics for the antibody while at the same time reducing contact time between Dynabeads® and the sample, thus minimizing non-specific binding. The Indirect Approach is also recommended when working with protein/nucleic acid complexes, e.g. in ChIP.

Incubation time

Increasing incubation times during immunoprecipitation can improve yield when working with low affinity antibodies. An incubation time of only 10 minutes is sufficient for most applications. Increasing the incubation time to 20-120 minutes can increase yield. Note that non-specific binding may increase with increasing incubation times.

Incubation temperature

For sensitive proteins, to avoid protein complex dissociation and minimize enzymatic activity e.g. when studying phosphorylation events, the isolation protocol including elution (2.5.B) may be run at 4°C.

Storage of Dynabeads®-Ab complex

For storage of Ab-conjugated Dynabeads®, we recommend using PBS (pH 7.4) with 0.01-0.1 % Tween-20 to prevent aggregation.

Storage of isolated protein

If the immunoprecipitated protein is to be stored, we recommend freezing the Dynabeads®-Ab-Ag complex after adding the elution buffer and sample buffer (step A.19). For analysis of the sample, thaw and continue the elution protocol from (step A.20).

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100.01D_02D.indd Rev 006 5-May-2009