The Seize X Immunoprecipitation Kit offers an excellent solution for immunopurifying proteins or complexes without co-elution of the capturing antibody. This advantage makes samples ready-to-use for further analysis and prevents contamination of western blot results with immunoglobulin heavy chains. Several options are offered for (i) immobilizing the customer's antibodies (Protein A or G coated beads) and (ii) preparing crude samples containing the protein of interest (bacterial, yeast or mammalian immunoprecipitation kit). After one hour of incubation and some quick spins in a benchtop microcentrifuge, immunopurified samples are eluted in harsh (low pH) or mild conditions (ionic strength) in hundreds of microliters of buffers and are then ready for further analysis. Moreover, the beads coated with the covalently immobilized antibodies are re-usable up to 10 times depending on the stability of the antibodies in the elution conditions.
Except for antibodies, the kit contains all reagents, buffers and tubes for 40 reactions (10 reactions for 4 immobilized antibodies: identical or different). A precise protocol and a 5x SDS-PAGE loading buffer are also included in the pack.
As in all immunoprecipitation protocols, the quantity of antibodies required is high: between 50 and 500 micrograms. However, cross-linking the antibody to the protein A or protein G beads allows the researcher to re-use the same beads for multiple assays (up to 10x) and consequently, to reduce cost by the same factor.
The SeizeX Immunoprecipitation Kit has been developed for relatively small volumes of sample, from 100 microlitres to few millilitres.
Thanks to the HandeeTM Spin cup columns and the microcentrifuge tubes, wash steps, elution, and re-equilibration of the resin take just a few minutes. For SDS-PAGE analysis, a 5x non-reducing loading buffer is also provided.
In the laboratory, we employ the SeizeX Immunoprecipitation Kit for concentrating HA-tagged proteins from cell extracts using immobilized monoclonal anti-HA antibodies. After an extra chemical precipitation step to collect the proteins in an adequate volume for gel loading (20ul), our samples are separated by SDS-PAGE and revealed by western blot with the same antibody as the one immobilized on beads, and without hindrance by IgG chains.
Thus, Pierce's kit can be employed for several applications such as small immupurifications of proteins; immunoprecipitations and co-immunoprecipitations of proteins or complexes, competition assays (epitope mapping, multimolecular protein interactions, etc.), optimization of immunopurification protocols before scale-up, screening of protein expression, even clarification of specific proteins in samples.
Researchers working on protein-protein interactions or protein immunopurifications will appreciate the ease-of-use of this kit and the options offered to prepare their samples from different sources (bacteria, yeast, mammalian cells), to immobilize antibodies on beads (covalently or not), and to analyze the eluted samples (in reducing or non-reducing conditions). In addition to oriented antibodies, versatile applications and conditions, the indisputable advantages of the SeizeX Immunoprecipitation Kit are the absence of immunoglobulin contamination in immunoprecipitated samples, and the possibility of re-using the antibody-coated beads several times. This kit is a useful tool for experts or untrained researchers who wish to save precious antibodies, time and money in their immnuprecipitation assays.
Christophe Moreau, Ph.D.
Post-Doctoral Fellow
Laboratoire de Biophysique Moleculaire et Cellulaire UMR5090 CNRS
CEA-Grenoble
France