RIP (radio-immune precipitation)

Radio-immune precipitation (RIP) can take over where a Western blot will fail you. Western blots let you know how much protein has accumulated in a sample. If you are more interested in the rate of synthesis of protein in a cell, or if your protein degrades too quickly to be detected by a Western blot, then RIP is definitely a technique you'll want to know about. RIP also detects protein-protein interaction, while Western blotting can't.

This chart should help illustrate the differences between RIP and Western blotting.

Let's look at this technique in greater detail.

1. Begin by radioactively labeling your cells.

You have the following choices:
• [35S] labels methionine—high energy, high specificity
• [3H] labels amino acids—low energy
• [14C] labels amino acids—low specific activity

2. Extract-release your protein mixture. You'll need to make antibodies against one protein, then incubate your protein in them. This will give you an antibody-protein complex, but it will also leave you with some free antibodies floating around. So you'll need to purify your antibody-protein complexes.

3. Use the bacteria staphylococcus aureus in excess to purify your protein. Before incubating your protein in staph aureus, fix it with formaldehyde or glutaraldehyde to kill the staph aureus, since it's a pathogen. The formaldehyde or glutaraldehyde will crosslink all of the proteins in the staph aureus cell. Staph aureus has on its surface a protein that binds to the tail of antibodies (to the Fc portion of the Ig molecule). That means it binds both free antibodies and your radiolabeled antibody-protein complexes.

4. Centrifuge the solution to get a precipitate. Discard the solution and save the pellet. Elute the antibody-protein complexes by boiling them in SDS sample buffer. This denatures the proteins and removes the antibodies from the staph aureus and the radiolabled proteins.

5. Run this on an SDS gel. Perform an autoradiograph and develop the x-ray film. You'll see a dark spot on the film for whatever protein was bound by the antibody.