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
Although an antibody-conjugated-bead approach is shown and the narrator specifically mentions 280 micron beads, better results are frequently obtained with smaller beads that can be more densely derivatized.
If your antigen-specific primary antibody doesn’t work when attached to a bead, try a two-step approach using your primary by itself in your sample, followed by capture with a secondary antibody (or Protein A/G) on the beads.
Optimum incubation time will depend on your antibody’s affinity.
I’ve never used sepharose resin for IP; pickled staph with either a monoclonal or polyclonal IgG primary usually works very well.
Great advice Shannon… thanks!
Magnetic racks are a rip off. If you are switching to magnetic beads, most companies will give you a free rack or two if you buy their beads. The more you by, the better chance of getting a free rack. Also ask manufacturers for a sample before you buy. Not all beads are created equal.
Magnetic racks are also easy to build, just buy the strongest possible magnets if you can’t get one for free.
I also agree that you should use the smallest possible magnetic particles. If at all possible use a two step IP protocol, they generally work the best despite the hassle. It is easier to agitate the mixture and keep it homogeneous and get quantitative IPs to work with a two step protocol.
Great shout Martini. Magnetic racks should be pretty easy to make. If anyone has made one and would like to contribute a post about exactly how they did it, drop me an email (nick at bitesizebio… you know the rest).
I’ve found that old laptop hard drives are pretty strong and inexpensive. All that is required is a bit of time and a star-shaped screwdriver. I use them in magnetic separations fairly frequently and they work quite well.
I have just posted a whitepaper about this very topic.
It discusses the technology shift for immunoprecipitation and how magnetic beads offer a more favorable technology than agarose or Sepharose slurry for IP by enabling rapid protein isolation, obtaining high signal-to-noise ratio and enabling reproducible results.
http://find.lifetechnologies.com/ip-whitepaper/blg
User beware: The yield and capacity of magnetic beads is much much lower than the traditional agarose/sepharose beads. So, the cost per mg/antibody bound (and therefore, cost/ug of target pulled down) is much higher than the traditional agarose IP beads.
The magnetic beads do have the advantages of faster washing, lower background, less void volume, and ease of preparing many samples at the same time. And, if you want to, you can automate IPs with magnetic beads.
If you’re looking for a co-IP, you may need the capacity of the agarose beads.
Cheers;
~Mike