Tap-tag Fusion Protein Purification

  1. Grow 2L of culture to an O.D.(600) = 1.5, which is approximately 15 g cells.

  2. Harvest cells by centrifugation at 4000 rpm for 10 minutes at 4oC.

  3. Wash cells with 500 ml cold distilled H2O.

  4. Resuspend cells in 30 ml cold distilled H2O, transfer to 50 ml falcon tube.

  5. Harvest cells by centrifugation at 3000 rpm for 5minutes at 4oC.

  6. Resuspend cells in 25 ml TTT, and harvest by centrifugation at 2000 rpm for 5 minutes at 4oC.

  7. Decant supernatant and freeze pellet in liquid nitrogen. Store at -80oC until ready for next step.

  8. Pre-cool coffee mill with dry ice (~80 ml). Run ~1minute. Discard dry ice just before use.

  9. Get 1 volume dry ice (~ 15 g) and place in mill.

  10. Transfer pellet to weigh boat (cover in foil and hammer first!), then place in mill.

  11. Run 90 seconds, stop after 30 seconds and mix each time.

  12. Transfer to lid, scrape out into pre-cooled cold beaker on ice, add 1 volume TAP-1 buffer, and let thaw on ice.

  13. Transfer to 50 ml conical tube, and spin at 4000 rpm for 10 minutes at 4oC.

  14. Transfer supernatant to cold SW41Ti tubes (14x89, 12 ml), and fill up to top with TAP-1.

  15. Spin in Beckman SW41Ti at 27000 rpm for 90minutes at 4oC.

  16. Add glycerol to 20% to the supernatant (1/5 vol).

  17. Freeze in liquid nitrogen and store at Ð80oC until ready for next step.

  18. Prepare beads:

    1. Wash 200 ul IgG agarose bead suspension with 5 ml of IPP150 in 15 ml conical tube.

    2. Wash 200 ul calmodulin bead suspension with 5 ml of IPP150/CalmB in 15 ml conical tube.

  19. Thaw extract, and spin in Beckman SW41Ti at 27000 rpm for 30minutes at 4oC.

  20. Add 50 ul 2 M Tris pH 8.0 ( final concentration is 10 mM) and 100 ul 10% NP-40 ( final concentration is 0.1%)..

  21. Rotate supernatant with IgG beads for 2 hours at 4oC in tube.

  22. Spin supernatant at 1800 rpm for 5 minutes at 4oC.

  23. Wash twice with 10 ml IPP150. Transfer to column.

  24. Wash column with 10 ml IPP150.

  25. Wash column with 10 ml TEV cleavage buffer.

  26. Close bottom of column, and add 1 ml TEV cleavage buffer with 100 U TEV enzyme (10 ul) .

  27. Close top of column, and rotate for 2 hours (or longer) at 16oC.

  28. Remove top and bottom plug on column, and recover eluate by gravity flow.

  29. Elute remaining stuff with 200 ul TEV cleavage buffer.

  30. To the 1.2 ml TEV eluate, add 3 ml CalmB buffer and 3 ul 1M CaCl2.

  31. Transfer to column containing washed calmodulin beads.

  32. Rotate column for 1 hr at 4oC.

  33. After binding, drain column by gravity flow.

  34. Wash column with 30 ml IPP150/CalmB.

  35. Elute 10 fractions of 200ul with IPP150/CalmElution buffer by adding 200ul aliquots, incubating 5 minutes, and collecting (also from cap). Repeat the 200 ul elution 10 times.

    Note that protease inhibitors are added from stocks to give 50 fold dilution.
    Example: 20 ul of stock per 1ml buffer.

    Use IMMEDIATELY after adding protease inhibitors.


    TAP-1 BUFFER
    (25 mM HEPES, pH 7.6, 200 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.5 mM EGTA, 1 mM DTT)

    TO MAKE 40 ml:

    • 1 ml 1M HEPES, pH 7.6 stock solution
    • 2.67 ml 3 M KCl stock solution
    • 80 ul 1 M MgCl2 stock solution
    • 8 ul 0.5 M EDTA stock solution
    • 40 ul 0.5 M EGTA stock solution
    • 40 ul 1 M DTT stock solution
    • Add four tablet sEDTA-free protease inhibitor
    • distilled H2O to 40 ml

    IPP150 BUFFER
    (10 mM Tris, pH 8.0, 150 mM NaCl, 10% NP40)

    TO MAKE 100 ml:

    • 1 ml 1M Tris, pH 8.0 stock solution
    • 3 ml 5M NaCl stock solution
    • 1 ml 10% NP40 stock solution
    • distilled H2O to 100 ml

    TEV cleavage BUFFER
    (10 mM Tris pH 8.0 150 mM NaCl, 10% NP40, 0.5 mM EDTA, 1 mM DTT)

    TO MAKE 30 ml:

    • 250 ul 1M Tris, pH 8.0 stock solution
    • 900 ul 5 M NaCl stock solution
    • 300 ul 10% NP40 stock solution
    • 30 ul 0.5 M EDTA stock solution
    • 30 ul 1 M DTT stock solution
    • distilled H2O to 30 ml

    IPP150/CalmB BUFFER
    (10 mM Tris, pH 8.0, 150 mM NaCl, 1 mM MgOAc, 2 mM CaCl2, 1 mM imidazole)

    TO MAKE 100 ml:

    • 1 ml 1 M Tris, pH 8.0 stock solution
    • 3 ml 5 M NaCl stock solution
    • 100 ul 1 M MgOAc stock solution
    • 200 ul 1 M CaCl2 stock solution
    • 50 ul 2 M imidazole stock solution
    • distilled H2O to 100 ml

    IPP150 Calm/Elution BUFFER
    (10 mM Tris, pH 8.0, 150 mM NaCl, 1 mM MgOAc, 2 mM CaCl2, 1 mM imidazole, 10 mM beta-mercapto-ethanol)

    TO MAKE 100 ml:

    • 1 ml 1M Tris, pH 8.0 stock solution
    • 3 ml 5 M NaCl stock solution
    • 100 ul 1 M MgOAc stock solution
    • 200 ul 1 M CaCl2 stock solution
    • 50 ul 2 M imidazole stock solution
    • 70 ul 14.4 M beta-mercapto-ethanol stock solution
    • distilled H2O to 100 ml

    50X STOCK Protease Inhibitors (store at -20oC)

    PMSF (87 mg/ml)
    ([500mM] phenylmethylsulfonyl fluoride)

    TO MAKE 30 ml:  Dissolve 2.61 g PMSF in DMSO to equal a final volume of 30 ml.

    LEUPEPTIN AND PEPSTATIN (5 mg/ml)

    TO MAKE 10 ml:  Dissolve 50 mg LEUPEPTIN or PEPSTATIN in DMSO to equal a final volume of 10 ml.

    TPCK (5 mg/ml)
    (tosylphenylalanine chloromethyl ketone)

    TO MAKE 10 ml:  Dissolve 50 mg TPCK in DMSO to equal a final volume of 10 ml.


    * Note: The EDTA in the Lysis buffer is also a protease inhibitor.