Immunoprecipitation (IP) Protocol

Instructions

Immunoprecipitation (IP) is a method used to purify and enrich target proteins and is based upon antigen-antibody specific reactions. The antibody binds with target proteins in a supernatant or cell lysate. The antibody can then react with protein A/G, coupled sepharose or magnetic beads or a secondary antibody. Protein complexes are separated with centrifugation and buffer is used to wash the complexes. The complex is suspended in loading buffer and then boiled for 10 min. Centrifugation is performed again to collect the supernatant containing antibodies, target proteins and other proteins. These supernatant proteins can be analyzed with a WB.

IP diagram courtesy of Jain P and Bhatla S.

Solution and reagents

• RIPA buffer (10mM Tris-HCl pH7.6, 1mM EDTA, 0.1% SDS, 0.1% NaDOC, 1% TritonX-100)
• Protein inhibitors
• PBS (refer to WB protocol LINK)
• PBST (refer to WB protocol LINK)
• 0.2M Glycine-HCl (pH 1.5-2.5)
• Tris-HCl (pH 9.4)
• 6×sample loading buffer (refer to WB protocol)

Sample preparation

Cell lysate samples

1. Scrape the cells in ice cold PBS and gather into a tube. Centrifuge at 2500 RPM to collect cells.
2. Add ice cold RIPA buffer into the cells (10⁷ cells per 1mL), which contains protease inhibitors. Suspend the cells in the buffer and sonicate the cells with appropriate settings.
3. Keep the buffer sample solution on ice for 20-30 min.
4. Centrifuge for 20min at 12000 RPM 4℃. Transfer supernatant into a new tube and quantify protein concentration with a BCA assay. A fresh sample is recommended, but the sample can be frozen at -80℃.

Tissue samples

1. Obtain required tissue(s). Wash blood away.
2. Grind the tissueusing liquid nitrogen followed by ultrasound sonication. Then, follow steps above for cell lysate preparation.

Immunoprecipitation

1. 0.5-1mg total protein in a total cell lysate volume of 500μl can be used for an IP reaction. Transfer the 500μl of cell lysate to a new tube.
2. Add 3-5 μg of primary antibody and incubate at 4℃ overnight to form an antigen-antibody complex.
3. The next day, add 50μl of magnetic beads to the complex solution for 2-4 hours at 4℃.
4. Centrifuge by using 2500 RPM at 4℃ to collect the antigen-antibody bead complex.

Wash and Elution or Thermal denaturation

There are two different methods that can be used to wash magnetic beads: acid elution and thermal denaturation. Acid elution is suitable for proteins of any size and results in a lower background. Thermal denaturation is more convenient, however, boiling protein A or G in the supernatant can potentially cause interference with downstream results. 

Wash the antigen-antibody-bead complex with a series of buffers as described below:

1. 1ml RIPA buffer
2. 1ml PBST buffer
3. 1ml PBS buffer

Repeat buffer series wash twice and centrifuge by 2500 RPM at 4℃

Acid elution

1. Elute the pellet twice with 50μl of elution buffer (150 mM glycine-HCl, pH 1.5-2.5).
2. Add 2μl of Tris-HCl (pH 9.4) to neutralize the elution buffer.
3. Add 20μl 6×sample loading buffer and boil the solution

Thermal denaturation

1. Add sample loading buffer according to the volume of the antigen-antibody bead complex
2. Boil the antigen-antibody bead complex at 100℃ for 10 min.
3. Transfer the supernatant to a new tube.

Detection

Perform a western blot (WB) to detect IP results. Load the protein sample into the SDS-PAGE gel and refer to the WB protocol Light and heavy chain interference is common. If the target protein weight is close to 55kD, using a light chain secondary antibody is recommended. If the target protein weight is close to 25kD, a HRP-protein A/G secondary antibody is recommended.