E. coli extract for bead halo assay

E. coli extract for bead halo assay

Buffers:

STE: 10 mM Tris-HCl (pH 7.5, 4°C), 100 mM NaCl, and 1 mM EDTA.

Extraction buffer: 50 mM Tris-HCl (pH 8.0, 4°C), 500 mM NaCl, 1 mM EDTA, 10 mM 2-mercaptoethanol, and 0.5 mM PMSF.

Transport buffer: 20 mM HEPES­-KOH (pH 7.3), 110 mM KOAc, 2 mM MgOAc, 5 mM NaOAc, 0.5 mM EGTA, 2 mM DTT, and 1 μg/mL each of aprotinin, pepstatin A, and leupeptin.

Strain: BL21.

Vector: pQE80L (Qiagen).

Culture: 15 mL LB (Amp) at 37°C till OD₆₀₀=0.4

Induction: when OD₆₀₀=0.4, shift to 20°C, add 0.1 mM IPTG, and incubate at 20°C for 16 hr.

Harvest: chill the culture on ice, spin down the cells at 4°C, and remove supernatant. Suspend the cells in 1 mL ice-cold STE, spin down at 4°C, and remove the supernatant. Freeze the cell pellet in liquid N₂ and sore at -80°C.

Extraction:

Suspend the frozen cells in 0.3 mL ice-cold Extraction buffer containing 0.3 mg/mL lysozyme, and stand on ice for 20 min. Sonicate on ice using a small tip for 10 sec ×2 times (we use Handy Sonic UR-20P (TOMY) at power level “3”). Centrifuge at 20,000 g for 15 min at 4°C. Recover the supernatant and dialyze it against Transport buffer at 4°C for >6 hr. Centrifuge at 20,000 g for 15 min at 4°C. Recover the supernatant, aliquot, freeze in liquid N₂, and store at -80°C until use.

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