Sepharose beads are porous, which gives them a high surface area for interaction with proteins and allows them to hold a lot of liquid. This is perfect for the application that they were originally designed for: purifying milligrams of protein in columns.
When immunoprecipitation (IP) – a small-scale technique for pulling specific proteins out of solution using bead-bound antibodies – was first developed, we naturally used sepharose beads, since that is what we were used to, and we had them in the lab. But for IP, the porous structure cases a problem since:
- The antibodies get trapped inside the pores and are difficult to wash away, so extensive washing is needed to reduce background levels
- The washing steps for IP are done in microfuge tubes and liquid exchange is done by pipetting, so it is easy to accidentally lose sample in the washes
- Diffusion is slow, so long incubation steps are required
- Long incubation and extensive washing cause mechanical and proteolytic damage the proteins
- All of these variables mean that the results are not as reproducible as we’d like
Improving the immunoprecipitation protocol using magnetic beads
Recently, there has been a growing trend towards using magnetic beads, rather than sepharose down for IP as they solve all of the problems listed above. Magnetic beads have a defined diameter, are non-porous and are, (you’ve guessed it) magnetic. This means that:
- There are no hidden surfaces for the antibodies to stick to so background, and the required number of washing steps, is reduced
- Diffusion is faster, so incubation steps are reduced
- Less washing and faster incubation times mean less chance of proteolytic damage
- A magnet can be used to separate the pellet and supernatant, which reduces the chance of losing sample and makes everything a lot faster and amenable to automation
- There are less variables so the process is very reproducible
So it looks like we should be switching to using magnetic beads for IP. What do you think?
Source: A rather excellent video called Immunoprecipitation On Bench Paper – A Surprising Shift, posted by LifeTechnologiesCorp on YouTube