Solubility Assay of His-Tagged Variants

Our solubility assay was performed as previously reported^{22 (link)}. In brief, single colonies of BL21(DE3) cells (New England BioLabs) transformed with each 6X His-tagged variant construct were grown overnight (~18 h) at 37° in 2 ml of auto-induction media. An equal number of cells were harvested, washed (50 mM Tris, 150 mM NaCl, pH 7.5) once, and lysed in E. coli lysis buffer (Wash buffer, Cell Lytic B ® (Sigma), and 100 μM phenylmethylsulfonyl fluoride) for 10 min at room temperature. In all, 5 μL of total cell lysate was diluted in 25 μL of wash buffer and added to an equal volume of 2x Laemmli sample buffer before western blot. Soluble protein was collected from the supernatant after a 15,000 × g spin for 10 min and diluted in equal amounts of 2× sample buffer for western blot or serially diluted 1:2 for dot blot (1 μL). All samples were boiled for 1–2 mins, run on SDS/12% PAGE, transferred to nitrocellulose paper, and probed with 1:1000 anti-His-HRP antibody (Santa Cruz Biotechnology #sc-8036-HRP). Densitometry was performed using ImageJ (NIH) to quantify immunoblots. For dot blots, one representative row from each serially diluted dot blot was quantified (n ≥ 3). Blots were derived from the same experiment and processed in parallel.