Anti his hrp antibody

The SDS-PAGE and western blot were carried out according to prior methodology [36 (link)]. Briefly, Aag2 cells were lysed in RIPA buffer and 15–20 µg of each cell lysate or 4–6 µg of purified protein samples were resolved in SDS-PAGE gel and then transferred onto the nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were probed with the following primary antibodies: anti-His HRP antibody (Santa Cruz, Dallas, TX, USA, Cat. no. sc-8036-HRP, 1:5000), anti-actin HRP (C4) (Santa Cruz, Dallas, USA, Cat. no. sc-47778 HRP, 1:6000), anti-pADPr antibody (10H) (Santa Cruz, Dallas, USA, Cat. no. sc-56198, 1:3000 dilution), and anti-V5 tag antibody (Thermo Fisher Scientific, Waltham, MA, USA, Cat. no. R960-25, 1:5000) and anti-CHIKV E1 (in house raised in mice, 1:3000). The blots probed with anti-pADPr antibody, anti-CHIKV E1 sera, and anti-V5 tag antibody were incubated with anti-mice IgG HRP antibody (Novus Biologicals, Colorado, USA, Cat. no. NB7539, 1:6000 dilution), washed with PBST, and then visualized using the Bio-Rad ChemiDoc MP System (Bio-Rad Laboratories, Hercules, CA, USA) after brief exposure to a chemiluminescent substrate. The uncropped images are included in supplementary information Figure S1.

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.