Academic Papers
(AutoPure100, LtCas12a) Bo Zhou, et al. Purification and functional validation of LtCas12a protein. STAR Protocols. 2023.
2026-08-17 15:54
(AutoPure100, LtCas12a) Bo Zhou, et al. Purification and functional validation of LtCas12a protein.
STAR Protocols. 2023. doi: 10.1016/j.xpro.2023.102600.
Highlights
• Detailed protocol for a Cas12a ortholog with TTNA PAM
• Purification of LtCas12a via multiple chromatographic techniques
• Validation of LtCas12a biological characteristics by various in vitro assays
Ion exchange chromatography (IEC).
a. Equilibrate SP Sepharose HP cation exchange column (Cytiva, Marlborough, MA, USA) with 5–10 column volumes buffer A (20 mM Tris (pH 7.5), 250 mM NaCl, and 5% Glycerol).
b. Pump the protein solution into the column for purification with the flow rate of 2.5 mL/min using a pure protein purification system (Inscinstech, Unique AutoPure100).
Gel filtration chromatography.
a. Pooled the peak fractions from the ion exchange chromatography together.
b. Concentrate protein product to 2 mL via 100 kDa ultrafiltration tube and centrifugation (3000g, 4°C).
c. Equilibrate Superdex200 pg 16/200 gel filtration column (Cytiva, Marlborough, MA, USA) with 1.5 column volumes size exclusion chromatography (SEC) buffer (20 mM Tris (pH 7.5), 250 mM NaCl, and 2 mM β-Mercaptoethanol).
d. Use the protein purifier (Inscinstech, Unique AutoPure100) for further protein purification and inject the concentrated protein product into the gel filtration column.
Official Website
Social Media
