The µMACS technology from Miltenyi Biotec uses super-paramagnetic microbeads, which are conjugated to tag-specific antibodies in order to purify his-tagged proteins which have been expressed in prokaryotic or eukaryotic cells. The anti-his microbeads bind to the his-tagged proteins and are then retained on a µ column (included in the kit). The columns are placed in the magnetic field of a µMACS Separator (purchased separately). After washing, the target protein is eluted using the supplied elution buffer. The purified protein can then be directly analyzed by SDS-PAGE and/or Western blotting. The µ columns cannot be reused.
The Miltenyi µMACS HIS Isolation Kit was originally designed for the isolation of proteins from eukaryotic cells, but it works very well with bacterial cells (e.g. E. coli); I isolated only from E.coli. In my experiments, I pelleted 2 ml of induced E.coli expression-culture. Lysis of cells was accomplished using the supplied lysis buffer (1 ml) in combination with sonication (5 x 20 pulses). I have also tried cell lysis using BugBuster from Novagen, which worked very well. After lysis, the clarified supernatant was incubated with 50 ul anti-his microbeads for 30 minutes during which time binding takes place. The microbead-cell lysate solution is then applied to the µ column and the column is placed in the magnetic field. Up to 4 samples can be processed in parallel. The washing steps include rinsing with 2 different washing buffers. If desired, aliquots can be collected for potential SDS-PAGE analysis. The typical elution buffer contains DTT, SDS, EDTA and bromphenol blue (ready for use in SDS-PAGE); before applying the elution buffer on the column, it is pre-heated to 95°C; the protein is denatured by the elution buffer. Native protein elution is also possible by using a pH shift or by eluting the whole antigen-microbead complex.
The isolation procedure from lysis to elution takes only about 1.5 hours. The system is very flexible as the volume of cell culture and washing stringency can both be varied without problems; non-denaturing elution is also possible. In my experiments, more than 80% of whole recombinant protein could be extracted. The eluted proteins were pure enough for immunological experiments. Therefore, the Miltenyi µMACS technology is appropriate for fast and efficient purification of his-tagged proteins and can be recommended.
The (in my opinion) expensive µMACS Separator (magnet) and the MACS MultiStand can be used with further products from the Miltenyi portfolio and may hence be a good investment.