Immunoprecipitation (IP) is a frequently used method for purifying specific proteins from complex samples such as cell lysates or extracts by using specific antibodies. Traditional IP protocols use Protein A, Protein G, Protein A/G, or Protein L coupled to an insoluble resin, such as agarose beads, to capture an antigen:antibody complex in solution. The complex is then recovered by centrifugation and boiling the complex with some denaturing buffer (e.g. Laemmeli buffer). Limitations of traditional IP include sample handling and processing difficulties, the inability to release native antigen from the beads for functional assays, poor reproducibility and recovery due to multiple wash steps.
The Catch and Release v2.0 kit from Upstate/Millipore overcomes many of the above limitations of traditional IP. Due to the use of spin columns, the IP procedure is faster and easier to perform, and more reproducible. The kit also enables the elution of the antigen:antibody complex without denaturation which allows for additional downstream applications.
The kit comes in different sizes, for 5 and 50 IP procedures. The kit contains all the required materials like wash buffer, antibody capture affinity ligand, to ensure binding of the capturing antibody to the resin contained in the included spin columns. Also included are capture tubes and denaturing (beta-mercaptoethanol needs to added) and non-denaturing elution buffer. The included protocol booklet is very detailed, includes a protocol for an immunoprecipitation kinase assay, and a great technical support and troubleshooting section.
In my hands, the kit was used for various routine IPs with different capture antibodies. While using the kit, some downsides were recognized. The use of chicken antibodies or human antibodies is not recommended. In regards to the chicken antibodies, a traditional approach using agarose coupled to goat anti-chicken antibodies was used instead (Millipore 2185).
As for all methods using antibodies, the incubation time and the antibody concentration needs to be tested empirically by the user. The protocol is very easy to follow. The spin columns get centrifuged to remove the resin slurry buffer. After some washes the bottom of the spin column gets plugged using the snap-off bottom plug. A total of 500 ul of reaction volume containing the cell lysate, antibody, affinity ligand, and wash buffer are added to the spin column. The top of the column gets capped and the mixture is incubated. After the designated incubation period the fluid gets spun out, and the column is repeatedly washed. The proteins may then be eluted using either the denaturing buffer or the non-denaturing buffer to recover the proteins in their native form.
This is a great and easy to use IP kit, offering flexibility on the elution buffer front, and good reproducibility due to the use of spin columns and therefore, standardized wash and elution steps.