Peroxidase conjugated goat anti mouse igg h l

Hippocampal tissues were incubated in 1 ml RIPA lysis buffer for 30 min to extract total protein. The 20 μg protein lysate was applied for conducting sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then was proceeded the polyvinylidene difluoride membranes transferring. After sealing for 1 hr in the mixed solution of phosphate‐buffered solution (PBS), Tween‐20, and 5% nonfat milk, the membranes were incubated with the related primary antibody against STAT3 (Cell Signaling Technology 1:2,000) at 4°C overnight. After 3 rinses with PBS, the proper secondary antibodies of Peroxidase‐Conjugated Goat Anti‐Mouse IgG (H + L) (1:15,000 dilution, YEASEN, cat: 33201ES60) and ProteinFindTM Goat Anti‐Rabbit IgG (Transgen, cat: HS101‐01) were subsequently utilized to culture with above membranes for 60 min at room temperature following by 3 rinses with PBS. Protein was quantified using a scanning laser densitometer (Biomed Instruments Inc.), and the gray value of the bands was simultaneously analyzed. Protein level of STAT3 was expressed as arbitrary units after normalization with GAPDH protein expression.

Recombinant E. coli BL21 strains containing plasmid constructs pET28a-inaQn-TB-P, pET28a-inaQn-P, and pET-28a (negative control) were induced with IPTG, washed twice and then resuspended in PBS. Surface-expressed P proteins from induced BL21-pET28a-inaQn-TB-P (GII.4) were released from bacteria by thrombin digestion as described in the previous section. For SDS-PAGE, the IPTG-induced bacteria were dissolved in 2× SDS-PAGE loading buffer (100 mM, pH 6.8 Tris-HCl, 4% SDS, 20% Glycerol, 0.2% Bromophenol Blue, 2% DTT), while the thrombin-released P proteins were dissolved in 5× SDS-PAGE loading buffer (250 mM, pH 6.8 Tris-HCl, 10% SDS, 50% Glycerol, 0.5% Bromophenol Blue, 5% DTT). Each sample was boiled for 5 min, and 10.0 μL from each was loaded and separated in a 12% SDS-PAGE gel, followed by staining with Coomassie Blue R250 (Beyotime, Shanghai, China). Western Blotting was conducted as described in a previous report (Wang et al., 2008 (link)). The antibody against GII.4 HuNoV recombinant viral capsid protein (1: 5,000; Niu et al., 2015 (link)), and peroxidase-conjugated goat anti-mouse IgG (H+L, 1: 3,000; Yeasen, Shanghai, China) were used as primary and secondary antibodies in Western Blotting as described in our previous publication (Niu et al., 2015 (link)). The 3, 3′-diaminobenzidine (Fedbio, Wuhan, China) was used as chromogenic substrate.