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  • Protocol for Removal of IMAC Contaminating Proteins (C2529)

    Inroduction

    E. coli SlyD, ArnA, and Can (carbonic anhydrase) are tagged with the chitin binding domain (CBD). Accordingly, these proteins may be removed by incubating the E. coli lysate or the IMAC elution fractions with chitin beads (NEB #S6651) or chitin magnetic beads (NEB #E8036). Binding of CBD-tagged proteins to chitin resin is compatible in a wide range of buffer conditions. Pooled IMAC fractions may be directly mixed with buffer-equilibrated chitin beads and incubated for 5–30 minutes to remove CBD-tagged contaminants from the His-tagged target protein.

    The following procedure is recommended:
    Use 1 ml of chitin resin for each volume of lysate or IMAC pool corresponding to 1 gram of NiCo21(DE3) cell pellet. (or use 1 ml of chitin resin for every 100 ml of expression culture). Resuspend chitin slurry (stored in 20% ethanol) and transfer to a gravity flow column. Equilbrate chitin column with buffer similar or equivalent to the IMAC low imidazole buffer: (or use a buffer compatible with the downstream chromatography step). Seal bottom of chitin column and add cell lysate or IMAC fractions containing CBD-tagged contaminants. Seal top of column and mix by rocking for 5–30 minutes at 4°C. Elute void volume containing target protein by gravity flow and optionally add extra equilibration buffer to displace all buffer containing the target protein.

    (Alternatively, if using a mini-spin column incubate sample 5–30 minutes before centrifuging to elute the target protein).

    Analyze eluted protein by SDS-PAGE or Western blot to determine purity. Removal of CBD-tagged contaminants may be analyzed by Anti-CBD Monoclonal Antibody (NEB #E8034).