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Reagent Preparation
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A Purification under native conditions: - Native Lysis buffer: NaH2PO4 50 mM, NaCl 300 mM, Imidazole 10 mM,?pH 8
Optional: Add 1 tablet protease inhibitor cocktail to the Lysis Buffer.
Tip: Lysis Buffer contains 10 mM imidazole to prevent binding of untagged proteins. If His-tagged proteins do not bind under these conditions, reduce the imidazole concentration to 1-5 mM. - Native Wash buffer: NaH2PO4 50 mM, NaCl 300 mM, Imidazole 20 mM,?pH 8
- Native Elution buffer: NaH2PO4 50 mM, NaCl 300 mM, Imidazole 250- 500 mM,?pH 8
Additional chemicals required: Lysozyme, Benzonase? nuclease,
Optional: Protease inhibitor cocktail
B Purification under denaturing conditions: - Denaturing Lysis buffer: NaH2PO4 100 mM, Tris base 10 mM, Urea 8 M,?pH 8.0,
Optional: Benzonase? nuclease (e.g. Merck Milipore, #707464) - Denaturing Wash buffer: NaH2PO4 100 mM, Tris base 10 mM, Urea 8 M,?pH 6.3NaH2PO4 100 mM
- Denaturing Elution buffer: NaH2PO4 100 mM, Tris base 10 mM, Urea 8 M,?pH 4.5
Tip: If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
Note: Due to urea dissociation, adjust the?pH immediately before use.
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