PureProteome Magnetic Stand

Comparison of elution techniques for small-scale protein purification of FLAG® tag proteins using anti-FLAG® M2 magnetic beads.

PureProteome™ Protein A and G Magnetic beads provide a rapid and reproducible means to purify immunoglobulins (IgG) using the KingFisher Duo particle processor.

比较anti-FLAG® M2磁珠的小规模FLAG®标签蛋白纯化的不同洗脱方法。

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.