SpCas9-NLS contains a nuclear localization sequence for optimal activity. Full length recombinant S. pyogenes SpCas9-NLS (conserved hypothetical protein), fused to a C-terminal His-Tag, expressed in E. coli. Molecular Weight: ~160kD Assay Conditions: Cas9 activity was measured using DNA Cleavage assay. Active Cas9 enzyme, sgRNA or tracrRNA/crRNA, 5ul of 4X Cas9 reaction buffer (200 mM Tris-HCl, pH 7.9, 400 mM MgCl2, and 0.4 mg/ml Prionex (w/v 0.04%)) and nuclease-free water were combined to a final reaction volume of 20ul. The reaction was then incubated at room temperature for 20-60 minutes, followed by the addition of 20-500 ng of target DNA to the Cas9-gRNA complex to initiate the reaction. A subsequent incubation at 37C for 10-30 minutes. Cleavage of target DNA was confirmed by running a DNA agarose gel. Applications: Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling Storage and Stability: Aliquot to avoid repeated freezing and thawing and store at -70C. Aliquots are stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
