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GST_purification

By Rick Bryant,2014-10-30 03:51
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gst

Raught Lab 2008

    GST-tagged Protein Purification:

    1. Resuspend pellet in 50uL of ice cold 1xPBS per ml pellet (for 1L overnight liquid

    culture, pellet is approximately 5ml, but 40mL of PBS is used to resuspend the

    pellet).

    2. Disrupt using sonicator (4-6x 30sec bursts, with ~1min rest on ice in-between)

    AVOID frothing.

    3. Transfer the lysate to 40mL centrifuge tube.

    4. Add Triton X-100 to 2% (800uL for 40mL), ensure that the Triton X-100 is

    dissolved then incubate @ 4?C end-over-end for 30 minutes.

    5. Centrifuge at 12,000g for 10 min @ 4?C.

    6. While centrifuging wash beads with PBS 3 times (2mL MagneGST beads for

    each 100mL of sonicate) {we normally use 520uL of MagneGST beads for 1L of

    culture}

    7. Transfer the centrifuged lysate to the tube containing the pre-washed beads.

    8. Incubate end-over-end @ 4?C for 2-3 hours.

    9. Wash beads with PBS 3 times.

    10.

    a) If storing beads, store in 1 bead volume of PBS supplemented with 5%

    glycerol @ 4?C.

    b) If eluting from beads, add 1 volume GST elution buffer, incubate end-over-

    end for 10 min @ 4?C. Collect supernatant. Repeat once more with the

    GST elution buffer and pool with previous supernatant. Repeat one last

    time with the supplemented GST elution buffer and pool with previous

    supernatants.

    c) If cleaving free of GST tag, see PreScission Cleavage protocol.

GST Elution Buffer Recipe:

    42 mg reduced dry glutathione in 40 ml of 50 mM TrisCL pH 8.0

    Filter through atleast 45um syringe filter and aliquot and store 6 months at -20 ?C

Supplemented GST elution buffer:

    Add of 150 mM NaCl, 5 μM CaCl (or MgCl) and 0.1% βME 22

    Raught Lab 2008

    PreScission? Protease Cleavage:

    1. Wash beads 3x with 2 beads volumes of PreScission? Protease cleavage buffer

    (1mL normally)

    2. Add 1 bead volume of cleavage solution [4% protease stock (2,000 units/ml) in

    cleavage buffer i.e. 20 μl protease in 480 μl buffer]

    3. Incubate end-over-end for 4 hours (usually overnight) @ 4?C.

    4. Collect the supernatant.

    5. Add 1 bead volume of cleavage buffer, and incubate end-over-end for 10 min.

    6. Pool supernatant together. Store at -80?C.

    PreScission? Protease Cleavage Buffer:

    Stock

    Solution Volume of Final

    Concentration Stock Concentration

    Tris pH 8.0 1M 2.5mL 50mM

    NaCl 5M 1.5mL 150mM

    EDTA 500mM 100uL 1mM

    DTT 1M 50uL 1mM

    NP-40 10% 50uL 0.01%

    MQ dHO to 50mL 2

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