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  • Preparation of Media and Solutions (E6901)

    Introduction

    The following are suggested media for cell culture, cell lysis and protein purification. They can be modified according to the specific properties of the target protein.

    LB broth (per liter)
    10 g tryptone
    5 g yeast extract
    10 g NaCl
    Adjust pH to 7.0 with NaOH

    Column Buffer
    20 mM Na-HEPES (or Tris-HCl), pH 8.5
    500 mM NaCl (or 50–1,000 mM NaCl)
    1 mM EDTA (optional)
    Nonionic detergents (0.1–0.5% Triton X-100 or 0.1–0.2% Tween 20) and protease inhibitors [e.g., PMSF (20 μM)] can also be included. For a target protein sensitive to oxidation, 1 mM of TCEP [tris-(2-carboxyethyl)phosphine] or TCCP [tris-(2-cyanoethyl)phosphine] may be used.

    Cleavage Buffer
    20 mM Na-HEPES (or Tris-HCI), pH 8.5
    500 mM NaCl (or 50-1,000 mM NaCl)
    50 mM DTT or β-mercaptoethanol or cysteine
    1 mM EDTA (optional)

    Stripping Solution
    0.3 M NaOH