Immunoprecipitation (IP) is a technique for capturing specific proteins from a complex solution via antibody-antigen affinity. In an IP, the goal is to pull out a specific protein. For a co-IP, instead of identifying individual proteins, the goal of the technique is to identify protein complexes. The phrase “pulling down” protein is commonly used to explain the process, but this idea is somewhat dated now that magnetic nanoparticles have begun to replace traditional centrifuge-based methods. The protein capture efficiency can be measured by IP input into SDS page and western blot analysis.
Immunoprecipitation accomplishes two main goals:
- Bind the target protein to an antibody on a solid support.
- This step requires incubation
- centrifugation (traditional)
- biomagnetic separation (modern)
Immunoprecipitation requires the attachment of antibodies to agarose-based porous resin columns (traditional), or to superparamagnetic nanoparticles (modern) . The solution is incubated with the chosen antibody-bound solid support, and antibody:protein complexes form.
Traditionally, resins are incorporated into single-use centrifuge tubes. Following incubation, the contaminating solution and unbound proteins are washed away before target proteins are eluted. This method requires a centrifuge and a technician to perform multiple wash steps.
Modern technology has introduced the superparamagnetic nanoparticle colloid as a free flowing, high surface area, recoverable solid support system for IP and co-IP. The movement of superparamagnetic nanoparticles in solution is controlled by an external magnetic field gradient. The magnetic nanoparticles are functionalized by the attachment of protein A/G or specific antibodies to bind target proteins in solution. The surface of these nanoparticles is designed to decrease non-specific binding in order to obtain the cleanest isolation possible. It is equally important to use a well-designed separation rack to maximize the speed and efficiency of the magnetic separation process. The use of magnetic nanoparticles in immunoprecipitation eliminates the need for a centrifuge, improves the speed of the process, and introduces the possibility of automation.
IP input into SDS page and Western Blot analysis
The goal of this step is to confirm that the immunpreciptation procedure indeed captured the target protein.
SDS-page is a method using gel electrophoresis to spatially separate denatured proteins by molecular weight. A portion of the cell isolate, prior to immunoprecipitation, is loaded into the ‘input’ column. A portion of the isolate, after immunoprecipitation, is loaded into the ‘ip input’ column. A third column is an isotype control. The proteins move through the gel down an electric field gradient and spread out into bands. Larger proteins travel at a slower speed than smaller proteins due to the drag force of the gel. When finished, the input lane will have many bands representing many different proteins, and the ip input lane will ideally have a single band representing the target protein.
The proteins are transferred from the SDS-page gel to a film for western blot analysis. The purpose of the western blot is to identify whether or not the immunoprecipitated protein is the target protein. A primary antibody to the target protein is applied to the western blot film transfer, followed by a dye-conjugated secondary antibody. If the ip input contained the target protein it will be visible on the western blot. A corresponding band will also be visible on the whole isolate input lane, but not in the isotype control.
The benefits of modern magnetic separators for immunoprecipitation protocols
Your immunoprecipitation might be for understanding the structure of a protein, its expression, it’s binding partners or any modifications it has. There are several advantages to using a modern magnetic separator that are adaptable to your experimental needs. Depending on which point you are starting your immunoprecipitation at, you will need a different size magnetic separator. Protocols in different labs will result in a variety of volumes that require separation. Modern magnetic separators range in size from milliliters to liters. No matter what size you use, the constant magnetic force will keep the separation process steady, fast, and efficient. You will be able to optimize this separation step with the magnetic separator monitoring technology. This will allow you to obtain the highest yield so you have more material to work with that is pure.