pET-Sac-Aβ(M1–42)

Protocol tips

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	For Aβ(M1–42) wild-type and mutant peptides: Wild-type or mutant plasmids were transformed into BL21 DE3 PLysS Star Ca2+-competent E. coli through heat shock method. The cell cultures were spread on LB agar plates containing carbenicillin (50 mg/L) and chloramphenicol (34 mg/L). Single colonies were picked to inoculate 5 mL of culture media for overnight culture. (A glycerol stock of BL21 DE3 PLysS Star Ca2+- competent E. coli bearing the plasmids was made, and the future expressions were started by inoculating culture media with an aliquot of the glycerol stock).

Protocol tips

For Aβ(M1–42) wild-type and mutant peptides: Wild-type or mutant plasmids were transformed into BL21 DE3 PLysS Star Ca2+-competent E. coli through heat shock method. The cell cultures were spread on LB agar plates containing carbenicillin (50 mg/L) and chloramphenicol (34 mg/L). Single colonies were picked to inoculate 5 mL of culture media for overnight culture. (A glycerol stock of BL21 DE3 PLysS Star Ca2+- competent E. coli bearing the plasmids was made, and the future expressions were started by inoculating culture media with an aliquot of the glycerol stock).

Publication protocol

All liquid cultures were performed in culture media (LB broth containing 50 mg/L carbenicillin and 34 mg/L chloramphenicol). For Aβ(M1–42) wild-type and mutant peptides: Wild-type or mutant plasmids were transformed into BL21 DE3 PLysS Star Ca2+-competent E. coli through heat shock method. The cell cultures were spread on LB agar plates containing carbenicillin (50 mg/L) and chloramphenicol (34 mg/L). Single colonies were picked to inoculate 5 mL of culture media for overnight culture. (A glycerol stock of BL21 DE3 PLysS Star Ca2+- competent E. coli bearing the plasmids was made, and the future expressions were started by inoculating culture media with an aliquot of the glycerol stock). The next day, all 5 mL of the overnight culture were used to inoculate 1 L of culture media. After inoculation, the culture was shaken at 225 rpm at 37 ˚C until the cell density reached an OD600 of approximately 0.45 (after around 3 h 45 min). Protein expression was then induced by the addition of isopropyl β-D-1- thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, and the cells were shaken at 225rpm at 37 °C for 4 h with IPTG. The cells were then harvested by centrifugation at 4000 rpm using a JA-10 rotor (2800 x g) at 4 ˚C for 25 min, and the cell pellets were then stored at -80°C.

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