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Immunoprecipitation (IP) protocol

General immunoprecipitation (IP) procedure covering lysate preparation, immunoprecipitation, and elution.
Last edited Wed 24 May 2023

Immunoprecipitation is a protein purification method that allows us to isolate a specific protein from the mixture using antigen-antibody interaction.

An antibody for the protein of interest is incubated with a cell extract enabling the antibody to bind to the protein in solution. The antibody/antigen complex is then pulled out of the sample using protein A/G-coupled agarose or magnetic beads. This isolates the protein of interest from the rest of the sample. The protein of interest can then be analyzed by western blot, mass spectrometry, direct ELISA, or other analytical techniques.

Stage 1 - Preparing the lysates

The first step is to lyse the cell or tissue samples in a suitable buffer to release proteins into the solution. We provide a non-denaturing lysis buffer (ab152163) suitable for isolating most proteins from cells or tissues.

Some proteins are more difficult to isolate than others, so you may need to create and optimize your own buffer. The ideal lysis buffer will minimize protein denaturation while releasing enough proteins from the sample.

Non-ionic detergents, such as NP-40 and Triton X-100, are less harsh than ionic detergents, such as SDS and sodium deoxycholate. Other variables that can affect the success of immunoprecipitation include salt concentration, divalent cation concentration, and pH.

Materials required

  • Suitable lysis buffer (example: ab152163, or develop your own)
  • Protease inhibitor cocktail (example: ab65621)
  • Phosphatase inhibitor cocktail (optional – example: ab201112) 
  • PBS

Steps

1

Prepare an appropriate lysis buffer for your protein.

Recommended use

Buffer

Recipe

Protein localized in membrane or cytoplasmic (mild lysis method)

NP-40 lysis buffer

150 mM NaCl, 1% NP-40, 50 mM Tris-HCl pH=8.0, 0.15% (w/v) BSA, 10% (v/v) glycerol, protease inhibitor cocktail and/or phosphatase inhibitors.

Protein localized in cytoplasmic or nuclear (harsh lysis method)

RIPA lysis buffer

50 mM Tris-HCl pH=8.0, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail and/or phosphatase inhibitors.

2

Add protease inhibitors to the buffer.

  • Include phosphatase inhibitors for phosphorylated proteins.
3

Isolate your cells and suspend them in a lysis buffer.

  • You should prepare a suspension with 300 µL of the lysis buffer added for 1–3 x107 cells or 600 µL of the buffer added for more than 3 x107 cells.
    • Some adherent cells may require enzymatic or mechanical detachment. We detach adherent cells with TrypLE, spin to remove the media, resuspend the pellet in PBS and spin two more times, each time resuspending the pellet in PBS to wash the cells, leaving a washed cell pellet at the end.
    • Suspension cells can be washed in PBS and spun down into a pellet before suspension in the ice-cold lysis buffer in the next step.
4

To lyse the cells, add ice-cold lysis buffer directly to the cell pellet and resuspend it.

  • Incubate the cells with lysis buffer on ice for 10 mins (without agitation).
  • Sonicate the lysate 3 times in ice-cold water.
5

Spin down the suspension to pellet insoluble contents.

  • Keep the supernatant – this is your lysate.
  • Centrifuge the suspension at 8,000 x g for 10 minutes at 4 °C. 
  • Place the supernatant in a fresh tube on ice.
6

Determine the protein concentration in your lysate using a Bradford or BCA assay.

  • Suppose the protein concentration at this stage is low, and your protein resides in the nucleus or mitochondria. In that case, you could consider fractionating your original sample to produce a more concentrated lysate. 
  • We offer cell fractionation kits for this purpose.
7

If not using immediately, snap freeze aliquots in liquid nitrogen and store at -80°C.

Stage 2 - Pre-clearing the lysates (optional)

Pre-clearing lysates is an additional optional step that can help to increase the purity of proteins obtained by IP. This step involves incubating the lysate with only beads or beads plus an isotype control, depending on the beads used, to precipitate unwanted proteins. One can normally skip this step when using high-quality beads without severe bead adsorption effect.

Pre-clearing the lysate can help reduce non-specific binding and reduce background. However, if the final detection of the protein is by western blotting, pre-clearing may not be necessary unless a contaminating protein interferes with the visualization of the protein of interest.

Here we provide a protocol example for pre-clearing the lysates using beads combined with an isotype control.

Materials required

  • Isotype control antibody
  • Your lysate
  • Protein A/G magnetic beads (example ab214286)
  • Magnetic rack

Steps

1

Prepare the beads and mix them with isotype control antibodies.

  • Follow the bead manufacturer’s advice to prepare the antibody-bead complexes.
2

Add a slurry of beads with isotype control antibodies to the lysate.

  • Add ~100 µL of beads per 1 mL of lysate.
  • Incubate for 10 – 30 mins at 4°C, with gentle agitation.
3

Remove the antibody-bead complexes.

  • Place the tube on a magnetic rack for ~1 min; the beads should be pulled by the magnet to the bottom of the tube.
  • Aspirate the solution, leaving the beads behind, and transfer this solution to a new tube.
4

Proceed to IP on your pre-cleared lysate.

Stage 3 - Immunoprecipitation and washing

Now we have lysed your cells and pre-cleared the lysate if necessary, so we’re ready to run the IP.

There are two main methods to immunoprecipitate proteins. The first approach is mixing the antibody with the lysate and then adding Protein A/G beads to the antibody-lysate complex. This method yields high purity of protein; however, the antibodies are also co-eluted with the protein of interest, which sometimes creates difficulties in western blot detection.

The second approach is preparing the antibody-bead complexes and then incubating them with your lysate to pull out the protein of interest. This method gives a lesser yield than the first one but avoids the problem of co-elution of antibodies.

We suggest you run IP according to the protocols below. If using an isotype control antibody, you can run the same procedure and compare this with your IP for the protein of interest when analyzing the results.

Materials required

  • Your lysate
  • Primary antibody
  • Isotype control antibody
  • Protein A/G magnetic beads (example: ab214286)
  • Magnetic rack

Steps

1

Incubate your lysate with antibodies at 4°C overnight, with gentle agitation.

2

Add the Protein A/G magnetic beads to the antibody-lysate complex.

  • Add ~100 µL of beads per 1 mL of lysate.
  • Incubate for 1–4 hours at 4°C, with gentle shaking.
3

Wash the beads three times with PBS or another wash buffer of your choice to remove unbound impurities.

  • Place the tube on a magnetic rack for ~1 min; the beads should be pulled by the magnet to the bottom of the tube.
  • Aspirate and discard the solution, keeping the beads in the tube.
  • Add ~ 1 mL of fresh lysis buffer to the beads and repeat the steps above.

Stage 4 - Elution

After washing, we’re ready to elute the protein from the beads. Elution can be done in various buffer conditions, including glycine (non-denaturing), Laemmli (denaturing), and urea buffers.

Of all these buffers, Laemmli buffer containing SDS is the harshest, as it will also elute non-covalently bound antibodies and antibody fragments along with the protein of interest. In contrast, glycine buffer gently elutes the protein with a reduced amount of eluted antibody.

This method produces a slightly less concentrated sample but keeps the protein in its native state. Since this is a non-denaturing buffer, the beads can also be re-used once the protocol is complete.

Materials required

  • 0.1 – 0.2 M Glycine, pH 2.6
  • Tris-HCl, pH 8.5
  • Your samples bound to beads

Steps

1

Add an equal volume of glycine to the beads.

  • Incubate for 10 mins at room temperature, with gentle agitation.
  • The low pH should separate the protein of interest from the antibody beads.
2

Separate the beads from the solution using a magnetic rack (for magnetic beads) or light centrifugation (for agarose beads).

  • For magnetic beads: place the tube on a magnetic rack for ~1 min; the beads should be pulled by the magnet to the bottom of the tube.
  • For agarose beads: spin down the tube at the manufacturer's recommended speed for 2 mins.
  • Aspirate the solution, leaving the beads behind, and transfer this solution to a new tube.
3

Neutralize the pH of the solution by adding Tris-HCl.

4

Optional: if not using immediately, aliquot samples, snap freeze, and store at -80°C.

5

Analyze the IP results using western blot or another technique of your choice.

  • Compare the results of IP performed with your capture antibody and isotype control antibody.