We found the Catch and Release® v2.0 to be a versatile new kit which made immunoprecipitation (IP) easier and simpler than traditional methods. Also advantageous was the availability of a trial-size: 5 columns plus all reagents, allowing evaluation of the system before purchasing the larger and more expensive kit.
The requirement for our laboratory was to find a method to immunoprecipitate low concentrations of enzymatically-active proteins from bacterial media concentrates. Traditional methods had caused difficulties due to their inability to release active, native-conformation proteins, and their inability to selectively recover only the protein of interest, due to inadequate systems for washing the resin. The design of the Catch and Release® system reduces and overcomes many of the limitations associated with traditional IP, including sample handling and processing difficulties, the inability to release native antigen for functional assays, poor reproducibility and poor recovery due to multiple wash steps. The spin-column format was also designed to make IP faster, simpler and more reproducible.
Catch and Release® Columns contain a proprietary resin in a microfuge-compatible column; the resin is secured by a screw cap top and a breakaway closure on the bottom. We found this aspect of the system most advantageous, as unlike the traditional methods, the ability to do all procedures within a single tube helped decrease the loss of resin and protein due to multiple manipulations. However, this does limit the volume of sample and antibody that can be used during the IP protocol to a total of 500 uL, which we found to be a severe limitation if a low affinity antibody and low concentration protein are under investigation.
We have successfully tested Catch and Release® on a number of proteins using both rabbit and mouse antibodies, and only 30 minutes of incubation was required. However, we found it advantageous to follow the manufactures recommendation that, if alternative conditions had previously been used in traditional IP procedures that the traditional method of incubation should be followed. It is recommended that researchers optimize the system and adjust binding times for each protein under investigation. Overall we found the ability to bind the antigen-antibody complex in as little as 30 minutes to be a significant time advantage compared with traditional IP methods.
We found the highlight of this system to be the quick and simple wash steps that ensured minimal contamination by non-specific proteins in the eluate. The main advantage being that there was no loss of resin, and consequently protein:antibody complex, in the washes. The quick spins and column format also removed the delays in waiting for the resin to settle, as is needed in traditional methods. Elution of the antigen:antibody complex is simple and can be done under either native or denaturing conditions using the buffers provided. We found this to be a significant advantage as it enabled us to immunoprecipitate active proteins that could then be directly used in other assays.
Department of Microbiology
Catch and Release® v2.0 Reversible Immunoprecipitation System From Upstate
Easy-to-use column immunoprecipitation system that overcomes some of the limitations of traditional immunoprecipitation.
Inability to batch purify large amounts of protein makes purification of low concentration proteins with a low affinity antibody difficult.
The Bottom Line
Easy-to-use method ideal for small scale immunoprecipitation.