PureProteome^™ NHS FlexiBind Magnetic Bead System

Immunoprecipitation (IP) is a powerful technique for proteomic screening, biomarker discovery, and signaling network elucidation. It is frequently used to enrich target proteins from complex samples such as cell lysates or extracts. Traditional IP protocols use Protein A, Protein G or a mixture of Protein A and G coupled to a solid support resin, such as agarose beads, to capture an antigen/antibody complex in solution. As the number of samples increase, the traditional, manual IP method can be time-consuming. Processing of multiple IP reactions in parallel can introduce complexity, variability and pipetting errors, which may affect reproducibility.

Traditionally, protein purification from E. coli consists of four distinct phases: harvest, bacterial cell lysis, lysate clarification and protein purification. Bacterial lysis typically requires several time-consuming, hands-on steps, such as freeze/thaw cycles and sonication. These harsh lysis techniques may negatively impact protein quality and contribute to sample-to-sample variability. To maintain protein activity and integrity, detergent-based lysis buffers are routinely used to avoid mechanical protein extraction methods. Regardless of the lysis method used, centrifugation is traditionally required to pellet unwanted cell debris and permit recovery of the clarified lysate. The final step, purification, is frequently performed using affinity media specific for expressed epitope tags. Agarose-based media have typically been used, either as a slurry in microcentrifuge tubes or packed into gravity-driven or spin columns. While easier to manipulate, columns are greatly affected by lysate consistency and carryover of cell debris, which can lead to clogging of the column frits.

Purification of recombinant proteins expressed in E.coli requires many time-consuming steps. To liberate the protein of interest, traditional bacterial lysis relies on the addition of lysozyme and a combination of sonication and repeated freeze/thaw cycles to break the bacterial cell wall. Disruption of the cell is accompanied by an increase in the viscosity of the suspension, due to the release of DNA. An endonuclease is added to digest the DNA, thus reducing the viscosity of the lysate. Finally, to render the lysate compatible with traditional purification methods, insoluble cell debris must be removed by centrifugation.

PureProteome™ NHS FlexiBind magnetic beads offer you flexibility in binding your target ligand. The kit contains everything you need, and offers high binding capacities (NHS density > 17 μmoles/mL of settled beads) with high specificity due to covalent linkages. NHS FlexiBind is perfect for applications involving targets that do not have affinity for common preconjugated magnetic beads.