To form Aβ42 oligomers and protofibrils, 15 μM Aβ42 monomers were vortexed for 30 seconds and then incubated at 4°C for two weeks as previously described (Ryan et al., 2010 (link)). Samples were centrifuged at 14,000 × g for 10 min at 4°C to eliminate insoluble mature fibrils. Oligomers and protofibrils were purified by size exclusion chromatography as previously described with a minor modification (Esparza et al., 2016 (link)). Briefly, 0.25 mL of sample was injected onto an Enrich SEC 650 column (Bio-Rad) attached to an NGC Quest Chromatography system (Bio-Rad), and eluted with PBS containing filtered 0.05% BSA at a flow rate of 0.5 mL/min. A total of 19 1 mL elution fractions were collected and resolved by PAGE. Fractions 8 and 9 were pooled as protofibrils whereas fractions 11 and 12 were pooled as oligomers. The concentration of Aβ42 oligomers and protofibrils were determined by ELISA (Thermo Fisher Scientific).
Ngc quest chromatography system
Manufactured by Bio-Rad
The NGC Quest Chromatography system is a highly versatile and automated liquid chromatography platform designed for a wide range of applications. It features precise flow control, reliable sample handling, and advanced data analysis capabilities to facilitate efficient purification and analysis of biomolecules.
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Lab products found in correlation
2 protocols using ngc quest chromatography system
1
Aβ42 Oligomers and Protofibrils Isolation
2
His-tag Protein Expression and Purification
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His-tag protein expression vectors were constructed using pTrcHis TA vector (Themo Fisher Scientific) according to the manufacturer’s instructions as previously described [3 (link)]. In brief, PCR amplified target genes were TA-cloned into the pTrcHis TA vector (Themo Fisher Scientific) and inserted DNA was verified by sequencing (Genewiz, South Plainfield, NJ, USA). Proteins were expressed in TOP10 competent cells (Themo Fisher Scientific) with IPTG (Themo Fisher Scientific) at 1 mM for 2.5 h at 37 °C, and were purified using a Nuvia IMAC Nickel-charged column (Bio-Rad) on an NGC Quest chromatography system (Bio-Rad). Proteins underwent dialysis against PBS using Slide-A-Lyzer Dialysis Cassettes, 10 K MWCO (Themo Fisher Scientific).
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