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Purification procedure under physiological conditions

The purification of Strep-tag®II fusion proteins is easy, straightforward and user-friendly. The complete procedure can be performed under nearly physiological conditions, e.g. in PBS buffer and for elution in PBS/2.5 mM desthiobiotin buffer:

Steps 1 + 2: The cell lysate is added to the column. Once the tagged protein has bound specifically to Strep-Tactin®, the host proteins are washed away rapidly with small amounts of physiological wash buffer (buffer W).

Step 3: Additional bound Strep-tag®II protein is gently eluted by adding wash buffer suuplemented with 2.5 mM desthiobiotin (buffer E), which specifically competes for the biotin binding pocket.
Since the buffer conditions during elution essentially remain unchanged, potentially unspecifically binding proteins (without Strep-tag®II) will not be eluted and, thus, will not contaminate the protein of interest. Next to the specific binding of Strep-tag®II to Strep-Tactin®, this is the second specificity conferring step of this purification procedure, yielding extremely high protein purity.

Steps 4: To regenerate the column, the yellow azo dye HABA (2- [4’-hydroxy-benzeneazo] benzoic acid) is added (buffer R) in excess to displace desthiobiotin from the binding pocket. Once HABA binds to the binding site, the color turns to red conveniently indicating the regeneration and activity status of the column.

Step 5: HABA can be removed simply by adding wash buffer. Once the red color has disappeared the column can be re-used. Strep-Tactin® resin can be regenerated and re-used atleast 3 to 5 times without loss of performance.