Millipore’s Catch and Release Immunoprecipitation (IP) Kit is an alternative to traditional IP protocols which predominantly utilize Protein A or Protein G coupled to agarose beads. The Catch and Release Kit overcomes many of the problems associated with traditional IP, such as poor reproducibility due to lengthy protocols and difficulty eluting the native antigen from the beads for functional assays.
Catch and Release utilizes a spin-column format which is designed to make IP a more streamlined process improving performance and increasing sample throughput. Catch and Release columns are loaded with a proprietary resin and have a screw cap top and a breakaway plug at the bottom. The system also contains an antibody capture affinity ligand which functions to bind the antigen:antibody complex to the resin. It is the antibody capture affinity ligand that allows the fast and simple elution of the antigen:antibody complex from the resin. The complex can be eluted in either a denatured or native form. 30 minutes of incubation with most antibodies is sufficient, and this results in a significant time savings when compared with traditional IP methods.
We have used this kit primarily to purify protein kinases from cell lysates. The procedure is quite straightforward and begins with preparation of the spin column. The first step is to remove the snap-off bottom plug and screw on cap and insert the spin column into a capture tube. The spin column is then centrifuged at 5000 rpm for 15-30 seconds to remove the resin slurry buffer. The column is then washed twice with the provided wash buffer. After washing the column, it is plugged with the snap-off plug. At this point, the following are added in a total volume of 500 ìl: 500 ìg of cell lysate, 1-4 ìg of a specific primary antibody, 10 ìl of antibody capture affinity ligand and sufficient Wash Buffer to bring the contents to the final volume. The column is then capped and incubated with mixing at room temperature for 30 minutes. After this incubation, the plug and cap are discarded and the column is centrifuged at 5000 rpm for 15-30 seconds. The column is washed another 3 times with 400 ìl of 1X Wash Buffer, spinning at 5000 rpm for 15-30 seconds per wash. At this juncture, the column is placed in a fresh Eppendorf tube and bound proteins are eluted from the column in either a denatured form by the addition of the elution denaturing buffer, or a native form using the non-denaturing elution buffer. Centrifuge the spin column at 5000 rpm and save the eluate for Western analysis or other assays.
This IP kit is faster and provides improved reproducibility over traditional IP methods. Some optimization may be required to determine the optimal quantity of antibody and the temperature and length of incubation with the antibody, but generally, in our hands and following the manufacturer’s recommendations, it has been successful.
Senior Research Scientist
Discovery Biology
Vitae Pharmaceuticals