Epitope tag antibodies are commonly used to study the interactions of the protein of interest with other proteins or nucleic acids. In the case of immunoprecipitation (IP), the protein of interest is expressed as a fusion protein with a tag cloned into either the N- or C-terminus. A tag-specific antibody that is immobilized onto a solid support is used to pulldown the protein of interest. Further confirmation is done by western blotting and/or other assay techniques.

Chromatin immunoprecipitation (ChIP) assays are performed to identify regions of the genome with which DNA-binding proteins, such as transcription factors and histones, associate. In ChIP assays, proteins bound to DNA are temporarily crosslinked and the DNA is sheared prior to cell lysis. The target proteins are immunoprecipitated along with the crosslinked nucleotide sequences which is then identified/analyzed by PCR, sequencing, microarrays, etc.
 

Materials

Preparation of the Dynabeads Protein A/Protein G Tag Antibody Complex

  1. Completely resuspend Dynabeads by pipetting or rotating on a roller for 5 minutes.
  1. Transfer 50 µL of Dynabeads to a tube, place on the magnet, and remove supernatant.
  1. Remove the tube from the magnet and resuspend Dynabeads in 200 µL of antibody binding and washing buffer containing your antibody of choice (typically 1-10 µg of antibody is used, the optimal amount will depend on the antibody used). Incubate for 10 minutes with rotation at room temperature.
  1. Place the tube on the magnet and remove supernatant.
  1. Remove the tube from the magnet and wash the Dynabeads-antibody complex by resuspending in 200 µL of antibody binding and washing buffer.

Protocol tips

Preparation of the Dynabeads-Tag Antibody complex involves incubation of Dynabeads-Protein A/G with the epitope tag antibody. The amount of antibody, Dynabeads, and incubation time required needs to be determined on a case-by-case basis. A good starting point is a 10-minute incubation with 1-10 µg of tag antibody and 50 µL of Dynabeads-Protein A/G.

Protocol tips

Specificity of Protein A or G to different Ig classes and subtypes varies and should be carefully considered before selecting an appropriate kit.

IPs should be done with a matched-isotype control antibody to rule out specificity-related issues.


Immunoprecipitation of the target antigen

  1. Place the tube on the magnet and remove supernatant.
  1. Add your antigen-containing sample (typically 100 - 1,000 µL) to the Dynabeads-antibody complex and gently resuspend by pipetting. Incubate for 10 minutes at room temperature with rotation.
  1. Place the tube on the magnet and transfer supernatant to a clean tube.
  1. Wash the Dynabeads-antibody-antigen complex 3 times, using 200 µL washing buffer for each wash. Mix gently by pipetting.
  1. Resuspend the Dynabeads-antibody-antigen complex in 100 µL washing buffer and transfer the suspension to a clean tube.
  1. Place the tube on the magnet and remove supernatant.

Protocol tips

For immunoprecipitation of the target antigen, the Dynabeads-Protein A/G-Tag Antibody complex is incubated with the lysate that contains the target fusion protein leading to formation of the Dynabeads-Protein A/G-Tag Antibody-Fusion Protein immune complex. While the amount of lysate used for this purpose and the incubation time needs to be calibrated on a case-by-case basis, the Dynabeads Protein A or G IP kit recommends 100-1,000 µL of lysate and a 10-minute incubation time as a starting point for optimization.

Protocol tips

A sufficient volume of untransfected and transfected lysates (~5-10% of input) should be kept aside to be used as controls in downstream analysis.


Elution of antibody/antigen complex

Note: For elution of the antibody/antigen complex, two alternative routes are available: denaturing or non-denaturing.

Denaturing

  1. Gently resuspend the Dynabeads-antibody-antigen complex in 20 µL of elution buffer.
  1. Add 10 µL NuPAGE LDS Sample Buffer or NuPAGE Reducing Agent. Mix and incubate for 10 minutes at 70°C.
  1. Place the tube on the magnet and load supernatant/sample onto a gel. Alternatively, the Dynabeads-antibody-antigen complex can be resuspended in the SDS sample buffer of your choice and heated as per your standard denaturing protocol prior to gel loading.


Non-denaturing

  1. Gently resuspend the Dynabeads-antibody-antigen complex in 20 µL elution buffer.
  1. Incubate 2 minutes at room temperature.
  1. Place the tube on the magnet and transfer supernatant/sample to a clean tube.
  1. Add 10 µL NuPAGE LDS Sample Buffer or NuPAGE Reducing Agent. Mix and incubate for 10 minutes at 70°C.


Analysis of immunoprecipitation by western blotting

The analysis of immunoprecipitation is usually done by western blot (use a protocol that is familiar or learn more in the Protein Gel Electrophoresis and Western Blotting Education Center).

However, one of the chief concerns regarding immunoprecipitation detection by western blot is the recognition of prominent antibody light (~25 kDa) and heavy chain (~55 kDa) bands. To circumvent this, it is recommended to use Clean-Blot IP Detection Reagent (HRP) instead for standard HRP conjugated secondary antibodies. This reagent is optimized for post-immunoprecipitation western blot detection of primary antibodies without interference from denatured IP antibody fragments.

Materials

Cross-linking of cells and chromatin preparation

Note: Cross-linking is essential for forming bonds between chromatin (DNA) and its associated proteins.
  1. Add 310 µL of 16% methanol-free formaldehyde to 2x107 cells (in 5 mL culture medium) and incubate at room temperature on a rocker for 12 minutes to start crosslinking.
  1. Stop crosslinking by adding 500 µL of 1.25 mM glycine (per 5 mL of culture medium) and incubate at room temperature for 5 minutes.
  1. Centrifuge cells at 2,500 RPM at 4°C for 5 minutes. Wash twice with 15 mL of 1X PBS.
  1. Resuspend cells in 1 mL of lysis buffer (50mM Tris, pH 8.0; 2mM EDTA, pH 8.0; 0.1% (v/v) NP40; and 10% (v/v) glycerol) containing 1X protease inhibitor cocktail and incubate on ice for 10 minutes.
  1. Transfer cells to 1.5 mL tube and centrifuge at 1,500 RPM for 5 minutes at 4°C. Then, resuspend the cells in 500 µL of nuclear resuspension buffer (50mM Tris, pH 8.0; 5mM EDTA, pH 8.0; and 1% (w/v) SDS) containing 1X protease inhibitor cocktail. Incubate on ice for 5 minutes, followed by sonication for 27 cycles on the ‘High’ setting on the Bioruptor.
  1. Centrifuge the sheared chromatin from step 5 at 13,000 g for 10 minutes. Discard the pellet, aliquot the supernatant, and store at -80°C or move directly on to the immunoprecipitation.

Protocol tips

  • Shear the chromatin to generate ~200–500 bp fragments for qPCR and ~100–300 bp fragments for massive parallel DNA sequencing analysis. In general, sonication conditions should be optimized for each sample type and instrument.
  • It is always preferable to use freshly prepared chromatin, however chromatin stored at -80°C could be used up to one week. Chromatin used beyond 1-week preparation time will not give optimal results.


Immunoprecipitation (IP)

  1. Take ~40 µL (equivalent to 1-2 million cells) of sheared chromatin and dilute with 9-fold volume (~360 µL) of ice-cold dilution buffer containing protease and phosphatase inhibitors. Store 10% of volume of the chromatin used per IP at -20°C. This chromatin will be used as a reference for ChIP-qPCR.
  1. Add the primary antibody (5 µg) to the diluted chromatin and rotate overnight at 4°C. For antibodies where the concentration is not mentioned, use 10 µL of antibody for each IP.

    In parallel, start a chromatin IP with equal amounts of rabbit or mouse IgG antibody as negative IP controls to measure non-specific binding. If using treated cells, start IP with equal amounts of primary antibody with untreated cells, as well.
  1. The next day, add 25 µL of Dynabeads (Protein A/G beads) to each sample and incubate for 1-2 hours with rotation at 4°C.
  1. Wash sequentially with 500 µL low-salt IP buffer 1 (twice), high-salt IP buffer 1 (twice), and IP buffer 2 (once). Rotate for 5 minutes at room temperature while washing.

Protocol tips

  • Do not forget to exchange bead storage buffer with dilution buffer at Step 3.
  • Do not forget to briefly spin the tube before putting it on the magnet.


Reverse cross-linking

Protocol tips

Make sure that the caps are tightly locked to prevent evaporation of water.

  1. After the final wash, completely remove the supernatant and resuspend the beads in 55 µL of complete reverse cross-linking buffer (53 µL reverse cross-linking buffer + 2 µL of proteinase K) and incubate at 55°C for 20 minutes.
  1. Briefly spin and place the tube on the magnet. Then, transfer the supernatant to a fresh tube and incubate further for 3-4 hours at 65°C.

    Do not forget to take out the stored reference samples and add complete reverse crosslinking buffer (53 µL reverse crosslinking buffer + 2 µL of Proteinase K) to a final volume of 55 µL (combined volume of reference & complete reverse crosslinked buffer) and incubate for 3-4 hours at 65°C along with immunoprecipitated chromatin samples.
  1. Purify the DNA with DNA purification module as per the instructions given below.


DNA purification using the PureLink PCR Purification Kit

  1. Add 220 µL of binding buffer B2 (with isopropanol) to the sample and mix well. Add B2 to a ratio of 4:1 (B2:sample).
  1. Pipet the sample into a spin column in a collection tube.
  1. Centrifuge the column at >10,000 x g for 1 minute. Discard the flow-through.
  1. Re-insert the column into the collection tube and add 650 µL wash buffer (W1) (with ethanol).
  1. Centrifuge the column at >10,000 x g for 1 minute. Discard the flow-through and place the column in the same collection tube.
  1. Centrifuge the column at maximum speed for 2-3 minutes.
  1. Place the column into a clean 1.7 mL elution tube.
  1. Add 100 µL elution buffer to the center of the column and incubate at room temperature for 1 minute.
  1. Centrifuge the column at maximum speed for 2 minutes.
  1. Now, the elution tube contains the purified DNA. Store at 4°C for immediate use or -20°C for long-term storage.


qPCR recipe and procedure

Protocol tips

Reaction Mixture for 1 sample is as follows:

  • PowerUp SYBR Green: 6 µL
  • ddH20: 2.8 µL
  • Primer (F+R): 1.2 µL
  • DNA: 2 µL
  • Total: 12 µL reaction mixture
  1. Depending upon the number of wells required for each sample, prepare a master mix. Add 10 µL of the master mix + 2 µL of DNA to the qPCR plate in duplicate (see Protocol tips).
  1. Seal the plate with optical adhesive film.
  1. Centrifuge for 1 minute at maximum speed and place the plate in the real time PCR system.
  1. Run the PCR machine with the desired settings.


Analysis: fold enrichment as calculated by signal over background

  1. Export the qPCR data to a spreadsheet program.
  1. Open the exported file. Average the replicate measurements for each IP reaction in a new column.
  1. The signal from each IP reaction is divided by the signal from the negative antibody control reaction. This represents the IP signal as the fold increase in signal relative to the background signal.
Immunoprecipitation (IP)

Materials

Preparation of the Dynabeads Protein A/Protein G Tag Antibody Complex

  1. Completely resuspend Dynabeads by pipetting or rotating on a roller for 5 minutes.
  1. Transfer 50 µL of Dynabeads to a tube, place on the magnet, and remove supernatant.
  1. Remove the tube from the magnet and resuspend Dynabeads in 200 µL of antibody binding and washing buffer containing your antibody of choice (typically 1-10 µg of antibody is used, the optimal amount will depend on the antibody used). Incubate for 10 minutes with rotation at room temperature.
  1. Place the tube on the magnet and remove supernatant.
  1. Remove the tube from the magnet and wash the Dynabeads-antibody complex by resuspending in 200 µL of antibody binding and washing buffer.

Protocol tips

Preparation of the Dynabeads-Tag Antibody complex involves incubation of Dynabeads-Protein A/G with the epitope tag antibody. The amount of antibody, Dynabeads, and incubation time required needs to be determined on a case-by-case basis. A good starting point is a 10-minute incubation with 1-10 µg of tag antibody and 50 µL of Dynabeads-Protein A/G.

Protocol tips

Specificity of Protein A or G to different Ig classes and subtypes varies and should be carefully considered before selecting an appropriate kit.

IPs should be done with a matched-isotype control antibody to rule out specificity-related issues.


Immunoprecipitation of the target antigen

  1. Place the tube on the magnet and remove supernatant.
  1. Add your antigen-containing sample (typically 100 - 1,000 µL) to the Dynabeads-antibody complex and gently resuspend by pipetting. Incubate for 10 minutes at room temperature with rotation.
  1. Place the tube on the magnet and transfer supernatant to a clean tube.
  1. Wash the Dynabeads-antibody-antigen complex 3 times, using 200 µL washing buffer for each wash. Mix gently by pipetting.
  1. Resuspend the Dynabeads-antibody-antigen complex in 100 µL washing buffer and transfer the suspension to a clean tube.
  1. Place the tube on the magnet and remove supernatant.

Protocol tips

For immunoprecipitation of the target antigen, the Dynabeads-Protein A/G-Tag Antibody complex is incubated with the lysate that contains the target fusion protein leading to formation of the Dynabeads-Protein A/G-Tag Antibody-Fusion Protein immune complex. While the amount of lysate used for this purpose and the incubation time needs to be calibrated on a case-by-case basis, the Dynabeads Protein A or G IP kit recommends 100-1,000 µL of lysate and a 10-minute incubation time as a starting point for optimization.

Protocol tips

A sufficient volume of untransfected and transfected lysates (~5-10% of input) should be kept aside to be used as controls in downstream analysis.


Elution of antibody/antigen complex

Note: For elution of the antibody/antigen complex, two alternative routes are available: denaturing or non-denaturing.

Denaturing

  1. Gently resuspend the Dynabeads-antibody-antigen complex in 20 µL of elution buffer.
  1. Add 10 µL NuPAGE LDS Sample Buffer or NuPAGE Reducing Agent. Mix and incubate for 10 minutes at 70°C.
  1. Place the tube on the magnet and load supernatant/sample onto a gel. Alternatively, the Dynabeads-antibody-antigen complex can be resuspended in the SDS sample buffer of your choice and heated as per your standard denaturing protocol prior to gel loading.


Non-denaturing

  1. Gently resuspend the Dynabeads-antibody-antigen complex in 20 µL elution buffer.
  1. Incubate 2 minutes at room temperature.
  1. Place the tube on the magnet and transfer supernatant/sample to a clean tube.
  1. Add 10 µL NuPAGE LDS Sample Buffer or NuPAGE Reducing Agent. Mix and incubate for 10 minutes at 70°C.


Analysis of immunoprecipitation by western blotting

The analysis of immunoprecipitation is usually done by western blot (use a protocol that is familiar or learn more in the Protein Gel Electrophoresis and Western Blotting Education Center).

However, one of the chief concerns regarding immunoprecipitation detection by western blot is the recognition of prominent antibody light (~25 kDa) and heavy chain (~55 kDa) bands. To circumvent this, it is recommended to use Clean-Blot IP Detection Reagent (HRP) instead for standard HRP conjugated secondary antibodies. This reagent is optimized for post-immunoprecipitation western blot detection of primary antibodies without interference from denatured IP antibody fragments.

Chromatin Immunoprecipitation (ChIP)

Materials

Cross-linking of cells and chromatin preparation

Note: Cross-linking is essential for forming bonds between chromatin (DNA) and its associated proteins.
  1. Add 310 µL of 16% methanol-free formaldehyde to 2x107 cells (in 5 mL culture medium) and incubate at room temperature on a rocker for 12 minutes to start crosslinking.
  1. Stop crosslinking by adding 500 µL of 1.25 mM glycine (per 5 mL of culture medium) and incubate at room temperature for 5 minutes.
  1. Centrifuge cells at 2,500 RPM at 4°C for 5 minutes. Wash twice with 15 mL of 1X PBS.
  1. Resuspend cells in 1 mL of lysis buffer (50mM Tris, pH 8.0; 2mM EDTA, pH 8.0; 0.1% (v/v) NP40; and 10% (v/v) glycerol) containing 1X protease inhibitor cocktail and incubate on ice for 10 minutes.
  1. Transfer cells to 1.5 mL tube and centrifuge at 1,500 RPM for 5 minutes at 4°C. Then, resuspend the cells in 500 µL of nuclear resuspension buffer (50mM Tris, pH 8.0; 5mM EDTA, pH 8.0; and 1% (w/v) SDS) containing 1X protease inhibitor cocktail. Incubate on ice for 5 minutes, followed by sonication for 27 cycles on the ‘High’ setting on the Bioruptor.
  1. Centrifuge the sheared chromatin from step 5 at 13,000 g for 10 minutes. Discard the pellet, aliquot the supernatant, and store at -80°C or move directly on to the immunoprecipitation.

Protocol tips

  • Shear the chromatin to generate ~200–500 bp fragments for qPCR and ~100–300 bp fragments for massive parallel DNA sequencing analysis. In general, sonication conditions should be optimized for each sample type and instrument.
  • It is always preferable to use freshly prepared chromatin, however chromatin stored at -80°C could be used up to one week. Chromatin used beyond 1-week preparation time will not give optimal results.


Immunoprecipitation (IP)

  1. Take ~40 µL (equivalent to 1-2 million cells) of sheared chromatin and dilute with 9-fold volume (~360 µL) of ice-cold dilution buffer containing protease and phosphatase inhibitors. Store 10% of volume of the chromatin used per IP at -20°C. This chromatin will be used as a reference for ChIP-qPCR.
  1. Add the primary antibody (5 µg) to the diluted chromatin and rotate overnight at 4°C. For antibodies where the concentration is not mentioned, use 10 µL of antibody for each IP.

    In parallel, start a chromatin IP with equal amounts of rabbit or mouse IgG antibody as negative IP controls to measure non-specific binding. If using treated cells, start IP with equal amounts of primary antibody with untreated cells, as well.
  1. The next day, add 25 µL of Dynabeads (Protein A/G beads) to each sample and incubate for 1-2 hours with rotation at 4°C.
  1. Wash sequentially with 500 µL low-salt IP buffer 1 (twice), high-salt IP buffer 1 (twice), and IP buffer 2 (once). Rotate for 5 minutes at room temperature while washing.

Protocol tips

  • Do not forget to exchange bead storage buffer with dilution buffer at Step 3.
  • Do not forget to briefly spin the tube before putting it on the magnet.


Reverse cross-linking

Protocol tips

Make sure that the caps are tightly locked to prevent evaporation of water.

  1. After the final wash, completely remove the supernatant and resuspend the beads in 55 µL of complete reverse cross-linking buffer (53 µL reverse cross-linking buffer + 2 µL of proteinase K) and incubate at 55°C for 20 minutes.
  1. Briefly spin and place the tube on the magnet. Then, transfer the supernatant to a fresh tube and incubate further for 3-4 hours at 65°C.

    Do not forget to take out the stored reference samples and add complete reverse crosslinking buffer (53 µL reverse crosslinking buffer + 2 µL of Proteinase K) to a final volume of 55 µL (combined volume of reference & complete reverse crosslinked buffer) and incubate for 3-4 hours at 65°C along with immunoprecipitated chromatin samples.
  1. Purify the DNA with DNA purification module as per the instructions given below.


DNA purification using the PureLink PCR Purification Kit

  1. Add 220 µL of binding buffer B2 (with isopropanol) to the sample and mix well. Add B2 to a ratio of 4:1 (B2:sample).
  1. Pipet the sample into a spin column in a collection tube.
  1. Centrifuge the column at >10,000 x g for 1 minute. Discard the flow-through.
  1. Re-insert the column into the collection tube and add 650 µL wash buffer (W1) (with ethanol).
  1. Centrifuge the column at >10,000 x g for 1 minute. Discard the flow-through and place the column in the same collection tube.
  1. Centrifuge the column at maximum speed for 2-3 minutes.
  1. Place the column into a clean 1.7 mL elution tube.
  1. Add 100 µL elution buffer to the center of the column and incubate at room temperature for 1 minute.
  1. Centrifuge the column at maximum speed for 2 minutes.
  1. Now, the elution tube contains the purified DNA. Store at 4°C for immediate use or -20°C for long-term storage.


qPCR recipe and procedure

Protocol tips

Reaction Mixture for 1 sample is as follows:

  • PowerUp SYBR Green: 6 µL
  • ddH20: 2.8 µL
  • Primer (F+R): 1.2 µL
  • DNA: 2 µL
  • Total: 12 µL reaction mixture
  1. Depending upon the number of wells required for each sample, prepare a master mix. Add 10 µL of the master mix + 2 µL of DNA to the qPCR plate in duplicate (see Protocol tips).
  1. Seal the plate with optical adhesive film.
  1. Centrifuge for 1 minute at maximum speed and place the plate in the real time PCR system.
  1. Run the PCR machine with the desired settings.


Analysis: fold enrichment as calculated by signal over background

  1. Export the qPCR data to a spreadsheet program.
  1. Open the exported file. Average the replicate measurements for each IP reaction in a new column.
  1. The signal from each IP reaction is divided by the signal from the negative antibody control reaction. This represents the IP signal as the fold increase in signal relative to the background signal.

Resources

For Research Use Only. Not for use in diagnostic procedures.