Immunoprecipitation (IP) - 3. Elution, Analysis

Now, you have agarose beads with the target proteins. Depending on your purpose, you can process IP, co-IP or RNA-IP (RIP). For chromatin-IP (ChIP), you need to add more steps before cell lysis. For IP and co-IP, you will do western blotting. When you detect target protein, it’s called IP. If you detect target interacting protein, it will be co-IP.

For example, I pulled down TRF1 protein. When I used TRF1-antibody as a primary antibody in western blotting, it’s called IP. If I used PINX1-antibody as the primary antibody, it’s called co-IP.

Analyze method for each purpose:

• Protein: Western blot

• RNA: RT-PCR, RT-qPCR, RNA dot/slot blot

• DNA: PCR, qPCR, ChIP dot/slot blot

Protein evaluation

1. Add 20 ul of 2X SDS sample buffer to the protein-agarose beads pellet.

2. Boil at 95°C for 5-10 min. Note1. Place a heavy block on top of the ep tube. Boiling the sample will make the cap pop open. Note2. The boiled sample can be stored at -20°C for short term, -80°C for long term.

3. Process western blot. Note. When you load samples on SDS-PAGE gel, use loading tips so that you don’t get agarose beads.

RNA evaluation

1. At the last washing, divide each sample into two tubes. Note. One tube will be used for wester blotting to check protein expression level, the other one will be used for RNA evaluation.

2. Once you get the beads pellet, add 1 ml of TRIzol.

3. Process RNA isolation.

Analyze

Figure 1. Western blot result example of co-IP. Anti-target interacting protein-antibody has been used as a primary antibody.

1. Input control band: IP was not performed. Thus, target interacting protein is detected. Housekeeping genes such as β-actin or GAPDH can be detected.

2. Input target band: IP was not performed. Thus, target interacting protein is detected. Housekeeping genes such as β-actin or GAPDH can be detected.

3. IP control band: IP product pulled down by IgG. Nothing was pulled down but the antibody-agarose beads complex. Because of the antibody, you will see each band of heavy chain and light chain. See Secondary Antibody for IP. Housekeeping gene can’t be detected.

4. IP target band: IP product pulled down by target interacting-antibody. If the protein you are looking for was interacting with the target protein, the target interacting protein band is detected. But also, because of the antibody used for pulling down, heavy chain band and light chain band is detected. Housekeeping genes can’t be detected.

Secondary Antibody for IP.

When you eluted the pulled down beads pellet for western blot, the antibody is also eluted and denatured. Antibodies consist of two different chains. One is heavy chain, the other one is light chain. Thus, when you boil the sample, the antibody is denatured to heavy chain and light chain. As secondary antibodies detect these chains, you will see the bands of those two chains. If your protein size is not similar to either heavy chain size (50 kDa) or light chain size (25 kDa), you will have no problems. However, if it’s overlapped, you have to use secondary antibody designed for IP since they only detects native antibody.