Immunoprecipitation
Immunoprecipitation (ip) is a technique for capturing specific proteins via antibody-antigen affinity from a complex solution. A co-ip, instead of identifying individual proteins, is designed 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 ito SDS page and western blot analysis.
Immunoprecipitation Methods
Immunoprecipitation accomplishes two main goals:
- Bind the target protein to an antibody on a solid support.
- incubation
- Isolate the antibody:protein complex from solution
- 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.
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