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.
Not radioactive, so it's easier
Tells how much protein has accumulated in a cell. If the protein is rapidly degraded, Western blotting won't detect it. You'll need to use RIP.
Tells the rate of synthesis of a protein.
Cannot detect protein-protein interactions.
Can detect protein-protein interactions. Two bands will show on a RIP gel. (Western blotting will only show one band.)
Let's look at this technique in greater detail.
1. Begin by radioactively labeling your cells.
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.
Guruatma "Ji" Khalsa. (2010, April 12). RIP (radio-immune precipitation). ASU - Ask A Biologist. Retrieved December 6, 2023 from https://askabiologist.asu.edu/rip-radio-immune-precipitation
Guruatma "Ji" Khalsa. "RIP (radio-immune precipitation)". ASU - Ask A Biologist. 12 April, 2010. https://askabiologist.asu.edu/rip-radio-immune-precipitation
Guruatma "Ji" Khalsa. "RIP (radio-immune precipitation)". ASU - Ask A Biologist. 12 Apr 2010. ASU - Ask A Biologist, Web. 6 Dec 2023. https://askabiologist.asu.edu/rip-radio-immune-precipitation