Mouse anti 6 his antibody

NC strips blotted with SDS-PAGE-separated kaouthiagin were blocked with 5% skimmed milk before incubating with lysates of individual E. coli clones containing soluble HuscFvs. Lysate of normal HB2151 was used as negative inhibition control. After washing with TBST, each strip was immersed in a solution of 1 µg/mL of 6× His-r-hvWF. Mouse anti-6× His antibody (Abcam, Cambridge, MA, USA), goat anti-mouse IgG-AP-conjugate (Southern Biotech) and BCIP/NBT substrate (KPL) were used for detecting the r-hvWF on the strips. Non-inhibition control, i.e., direct binding of r-hvWF to the kaouthiagin, was included in the assay.

Overnight cultures of E. coli BL21(DE3) carrying pET28-6 × His-tet(X2), pET28-6 × His-tet(X3), pET28-6 × His-tet(X4) and the variants were diluted 1:100 into 5 mL LB broth containing 50 μg/mL kanamycin. Cells were grown to OD₆₀₀ = 0.6 at 37 °C and induced by adding 1 mM IPTG for 4 h at 30 °C. Cell was harvested by centrifugation at 13,000 rpm. Cell lysates were solubilized by boiling with SDS running buffer for 10 minutes and were subsequently separated by SDS-PAGE. Proteins were transferred to a PVDF membrane followed by blocking by skimmed milk for 1 h and incubated with mouse anti-6 × His antibody (ABCAM, USA) at 4 °C overnight. The goat anti-mouse antibody (ABCAM, USA) was used as the secondary antibody. The signal was generated by HRP substrate and detected by ChemiDoc Touch System (Bio-Rad, USA). Tet(X2) was used as a positive control on each protein gel and cells containing the empty vector was used as a negative control. The broad range anti-GADPH (ABCAM, USA) was used as loading control. Band intensities were quantified using ImageJ software.