Symptomatic leaves were harvested 11–13 dpi and blender-ground in 100 mM potassium phosphate buffer, pH 7.3–7.6, 100 mM NaCl, 0.04% (v/v) Triton ×100, 5 mM BME, and 0.1 mM PMSF (1:3 w/v). The homogenate was filtered through three cloth layers and centrifuged at 6,000 × g to remove cell debris and insoluble material. The soluble extract was subjected to a two-step purification process involving a first stage by Ni2+-sepharose affinity chromatography, and a second stage by Cation Exchange in a HiTrap SP Sepharose FF 5 ml (AKTA Xpress System -GE), according to Williams et al. (2016) (link). The absorbance (280 nm wavelength) peaks were analyzed on a 15% SDS-PAGE and by western blot (as described below). Purified NPC-2 protein was quantified by SDS-PAGE and UV spectrophotometry by Nanodrop.
Hitrap sp sepharose ff 5 ml
Manufactured by GE Healthcare
HiTrap SP Sepharose FF 5 ml is a prepacked, ready-to-use column for rapid and convenient ion exchange chromatography. It is designed for the purification of proteins, peptides, and other biomolecules.
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Lab products found in correlation
2 protocols using hitrap sp sepharose ff 5 ml
1
Purification and Quantification of NPC-2 Protein
2
Recombinant 15N-tau Protein Purification
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Competent Escherichia coli BL21(DE3) bacterial cells were transformed with the various PHEN2-VHH constructs. Recombinant E.coli cells produced proteins targeted to the periplasm after induction Recombinant 15 N-tau production was induced with 0.5 mM IPTG when the culture reached an OD600 of 0.8. 15 N-tau was first purified by heating the bacterial extract 15 min at 75°C. The resulting supernatant was next passed on a cation exchange chromatography column (Hitrap SP sepharose FF, 5mL, GE healthcare) equilibrated in 50 mM sodium phosphate buffer (NaPi) pH 6.5 and eluted with a NaCl gradient. tau pooled fractions were buffer-exchanged in 50 mM ammonium bicarbonate (Hiload 16/60 desalting column, GE Healthcare) for lyophilization 38 (link) .
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