The newly engineered Strep-Tactin®XT has an improved binding affinity to Twin-Strep-tag® and Strep-tag®II. Strep-Tactin®XT now enables new applications in the field of high throughput screening, batch purification, purification using denaturing conditions and protein interaction studies. It also provides higher protein yields compared to Strep-Tactin®. Simultaneously, Strep-Tactin®XT preserves all benefits of Strep-Tactin® for protein purification, e.g. high purity (> 95 %), physiological buffer conditions, mild elution, regeneration of the resin for re-use.
Get highly pure proteins with Strep-Tactin®XT Superflow resin
One of the key advantageous of the Strep-tag® system is to achieve unparalleled protein purity (> 95 %) after purification of recombinant proteins independent of the expression host. This makes is system advantegous to other affinity purification systems like His-tag.
This Strep-tag® system III with its new features makes the system suitable for additional applications:
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Step 1: The cell lysate / culture supernatant is applied on the column.
Step 2: Once the tagged protein has bound specifically to Strep-Tactin®XT the host proteins are instantly washed away with moderate amounts of physiological wash buffer (Buffer W).
Step 3: Bound Twin-Strep-tag® protein is gently eluted by Buffer BXT (wash buffer containing 50mM biotin) which specifically competes for the biotin binding pocket.
Step 4: Regeneration of the column is achieved by the application of 10mM NaOH. Sodium hydroxide removes the biotin from the binding pocket. NaOH can be removed simply by applying Buffer W. Strep-Tactin®XT resin can be regenerated and re-used 3 to 5 times without loss in performance.
Shown is the comparison of two randomly chosen protein, which were either fused to 6xHis-tag or Twin-Strep-tag®. Protein were purified via NiNTA resin, which was done under optimal His-tag purification conditions, or via Strep-Tactin®XT resin using physiological buffer conditions on the right side, respectively. Both proteins show low background when purified using the Strep-tag® system.
Improved performance of the 3rd generation Strep-tag® system
Strep-Tactin®XT allows the purification of higher protein yields compared to Strep-Tactin®. This was determined by purification of different target proteins on either Strep-Tactin® or Strep-Tactin®XT. The obtained protein yields were determined and compared in the graph below. Here, we found that Strep-Tactin®XT allows in average the purification of a 2-fold higher protein yield compared to Strep-Tactin®.
An advantage ot the new Strep-tag® system III is that it allows intensive washing without the loss in protein yield. This figure shows the purification of mCherry-Twin-Strep-tag® fusion protein via 1 ml Strep-Tactin® Superflow® resin and 1 ml Strep-Tactin®XT Superflow® resin. E. coli supernatants were prepared and applied onto the column. Then the columns were washed with 8 CV Buffer W in order to reach a constant absorption at A260/A280 nm. Then the target protein was eluted with biotin. Samples of the last wash fraction and the elution fraction were analysed on SDS-PAGE showing that some of the target protein was washed down from Strep-Tactin® Superflow® resin, whereas no protein was eluted from Strep-Tactin®XT Superflow® resin.
The elution fractions were further analysed in Western Blot, showing that the remaining bands are degradation products of mCherry.