This video demonstrates the cation exchange chromatography technique to isolate recombinant protein from the insect cell protein lysate.
Protocol
1. Protein purification Prepare buffers A–H, as seen in Table 1. Buffer solution Buffering agent (all 20 mM) pH NaCl (mM) Imidazole (mM) MgCl2 TCEP <…
Representative Results
Figure 1: SDS-PAGE gels of the purification process. (A) IMAC capture from lysate. FNTA/B are the dark bands migrating at ~48 kDa and the His6-MBP-tev-KRAS4b is the dark band migrating at ~67 kDa. M = protein molecular weight ladder; T = total protein; L = clarified lysate/column load; F = column flow through. Fractions are eluted with an increasing imidazole concentration gradient. (B</str…
Disclosures
The authors have nothing to disclose.
Materials
1.8 mL Safe-Lock Tubes, Natural
Eppendorf
22363204
11 mm Cl SS Interlocked Insert Autosampler Vials
Thermo Scientific
30211SS-1232
5427R Centrifuge
Eppendor
Acetonitrile, HPLC Grade
Fisher Chemical
A998-1 1L
Cation Exchange Chromatography (CEX) column
GE Healthcare Life Sciences
29018183
HiPrep SP Sepharose High Performance
Exactive Plus EMR Mass Spectrometer
Thermo Scientific
Gilson vials 7×14 mm Tubes
GE Healthcare
BR-1002-12
High speed/benchtop centrifuge
Thermo Fischer Scientific
05-112-114D
Capable of up to 4,000 xg
His6-Tobacco Etch Virus (TEV) protease
Addgene
92414
Purified as per Raran-Kurussi et al. (2017) Removal of Affinity Tags with TEV Protease. In: Burgess Brown N. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 1586. Humana Press, New York, NY
Protease Inhibitor Cocktail without EDTA or other chelators
Cation Exchange Chromatography: A Technique to Isolate Target Recombinant Protein from Insect Cell Lysate Based on Net Surface Charge. J. Vis. Exp. (Pending Publication), e21096, doi: (2023).