Hrp labeled igg secondary antibody

Protein content was quantified, then protein separation was completed through electrophoresis, followed by transfer on PVDF membranes. Afterwards, PVDF membranes were subjected to 2-h blocking using 5% defatted milk powder, overnight incubation using TBST-diluted primary antibodies (dilution, 1:800, v/v) under 4°C, and later incubation using TBST-diluted, HRP-labeled IgG secondary antibody (dilution, 1:2000, v/v; Abcam, Massachusetts, USA). Afterwards, the chemiluminescent immunoassay was conducted for protein blot detection, Image Pro-Plus 6.0 software was employed to analyze optical density, and GAPDH was used as internal control. Data were represented by optical density ratio of target protein to internal control protein.

The intestinal tissue proteins were extracted in line with ELISA assay. After protein quantification, the proteins were separated by electrophoresis and transferred onto the PVDF membrane. Later, the PVDF membrane was blocked with 5% defatted milk powder for 2 h, incubated with the TBST-diluted primary monoclonal antibodies (dilution, 1:300-1:500; Abcam, MA, USA) at 4° C overnight. Later, the membrane was further incubated with the HRP-labeled IgG secondary antibody (Abcam, MA, USA). The IgG was diluted with TBST at the volumetric ratio of 1:2000 for subsequent experiment. After incubation, the protein bands were detected with chemiluminiscence, and optical density (OD) was analyzed with the Image Pro-Plus 6.0 software, with GAPDH as the endogenous reference. The results were expressed as the OD ratio of target protein to endogenous protein.

Protein content was measured, then samples were subjected to electrophoresis for protein separation, followed by transfer on PVDF membrane. Thereafter, 5% defatted milk was added to block membranes for a 2-h period, prior to overnight incubation using TBST-diluted primary monoclonal antibodies (dilution, 1:300-1:500; Abcam, MA, USA) under 4^° C. Afterwards, HRP-labeled IgG secondary antibody (1:2000; Abcam, MA, USA) was further added for membrane incubation. Then, chemiluminescence was conducted to detect protein bands, while Image Pro-Plus 6.0 software was employed to analyze absorbance (OD), and GAPDH was an internal control. Results represented target protein-to-control protein OD ratio.