Millipore Corporation
17 Cherry Hill Road
Danvers, MA, 01822
Website: http://www.millipore.com
|
 |
Improving Immunoprecipitation
by Maria Wasienko
Introduction
Immunoprecipitation is a technique that is frequently used to purify specific proteins from complex samples such as cell lysates or extracts. Traditional immunoprecipitation protocols use Protein A or Protein G coupled to an insoluble resin, such as agarose beads, to capture an antigen:antibody complex in solution. The complex is then "precipitated" by centrifugation. The limitations of traditional immunoprecipitation include sample handling and processing difficulties; the inability to release native antigen from the beads for use in functional assays; and poor reproducibility and recovery due to multiple wash steps.
The Catch and Release Reversible Immunoprecipitation System (Millipore Corp., Billerica, MA) overcomes many of these limitations, and allows researchers to conduct immunoprecipitation without Protein A or G Agarose. Protein can be eluted in either native or denatured form, and the spin column format provides a simplified and accurate process.
How it works
Figure 1. The Catch and Release reversible immunoprecipitation protocol. Click to enlarge. |
Columns in the Catch and Release system contain a proprietary resin in a microcentrifuge-compatible spin column format. An Antibody Capture Affinity Ligand for binding the antigen:antibody complex is also provided, and serves as a tether between the complex and the resin. It is this feature that enables the fast and simple elution of the antigen:antibody complex from the resin in either a denatured or native form.
The system has been successfully tested on a number of proteins with both rabbit and mouse antibodies. As little as 30 minutes of incubation with most antibodies can provide the desired results.
System advantages
Using an all-inclusive kit for immunoprecipitation protocols offers significant savings in time and reagents as well as other benefits. The absence of Protein A or G Agarose provides less non-specific binding to antibodies, and the spin column format ensures ease-of-use and decreased sample loss. Traditional immunoprecipitation methods lacking a spin column can be difficult to operate and sometimes provide inconsistent results. Choice of elution type allows for isolating the protein in either the active state for use in kinase assays or the denatured form for SDS PAGE/western blot analysis. Without this ability in traditional immunoprecipitation, fractional assays may not be readily available.
Conclusion
Isolating a specific antigen from a complex protein mixture has proven to be an invaluable and routinely employed investigational tool. Using the Catch and Release kit to isolate the protein provides numerous benefits. The spin column format makes immunoprecipitation fast and reproducible, while at the same time providing high throughput processing of samples. The absence of Protein A or G agarose with the added ability to elute protein in either native or denatured form provides a simplified process.
About the author
Maria Wasienko, Product Manager, Millipore Bioscience Division. More information is available from the company at:
Millipore Corp.
800-645-5476
www.millipore.com
|
|
