Hitrap capto s column

Overexpression
plasmids were constructed by subcloning TEM-1 into a pET24 vector
(Life Technologies) with its native export signal sequence replaced
by the OmpA signal sequence to maximize export efficiency.^{21 (link)} Variants were constructed via site-directed
mutagenesis and verified by DNA sequencing. Plasmids were transformed
into BL21(DE3) cells (Life Technologies) for expression under a T7
promoter control. Cells were induced with 1 mM IPTG at OD = 0.6 and
grown at 18 °C for 15 h before harvesting.
TEM β-lactamases
were isolated from the periplasmic fraction using osmotic shock lysis:
Cells were resuspended in 30 mM Tris-Cl, pH 8, and 20% sucrose and
stirred for 10 min at room temperature. After centrifugation, the
pellet was resuspended in ice-cold 5 mM MgSO₄ and stirred
for 10 min at 4 °C. After centrifugation, the supernatant was
dialyzed against 20 mM sodium acetate, pH 5.5, and purified using
cation exchange chromatography (HiTrap Capto S column, GE Healthcare).
All variants eluted between 10 and 20% NaCl. Proteins were concentrated,
dialyzed in storage buffer (20 mM Tris, pH 8.0), and final concentrations
were measured in Edelhoch buffer using calculated extinction coefficients
based on the number of tryptophans, tyrosines, and disulfide bonds.^{22 (link)}

2N4R tau lacking the
endogenous cysteine residues (C291S and C322S) and the relevant cysteine
mutants (S262C and K311C), created by standard site-directed mutagenesis,
were expressed from a pet29b vector in BL21 Gold (DE3) cells (Agilent
Technologies). Cultures were grown to an OD600 of 0.6 and then induced
with 0.4 mM IPTG and left to express at 18 °C overnight. Cells
were harvested by centrifugation, resuspended in 50 mM MES (pH 6.5),
5 mM DTT, 0.1 mM PMSF, and lysed via sonication (1 min 30 s; 5 s on,
10 s off; 40% amplitude) on ice. The lysed mixture was centrifuged,
and tau was isolated via cation exchange using a Hitrap CaptoS column
(GE Healthcare LifeSciences, Little Chalfont, U.K). Fractions containing
tau as determined by gel electrophoresis were pooled and precipitated
by the addition of 20% (w/v) ammonium sulfate on ice overnight. The
precipitated protein was pelleted by centrifugation and then resuspended
in SSPE buffer containing 5 mM DTT. Pure tau was finally isolated
via size exclusion chromatography using a Superdex 200 Increase 10/300
GL column (GE Healthcare LifeSciences, Little Chalfont, U.K.) equilibrated
with the aforementioned SSPE buffer; only the purest fractions as
assessed by gel were kept for experiments.