The titers of the prepared polyclonal antisera were tested using indirect enzyme-linked immunosorbent assay (ELISA) methods as described in a previous report [20 (link)]. Western blot was used to detect the two prepared polyclonal antisera as described by Wang et al. [21 (link)]. Briefly, proteins extracted from chicken cecal tissues using RIPA Lysis Buffer (Beyotime Biotechnology) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. The membranes were probed with prepared rabbit anti-ChNLRP3 and anti-ChTLR15 polyclonal antisera. The membranes were washed twice with TTBS (0.1% Tween 20, 50 mmol/L Tris–HCl, 150 mmol/l NaCl, pH 7.5) and reacted with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Sigma, USA). After washing, the target immunoreactive bands were visualized by an ECL chemiluminescence detection kit (Sangon Biotech Co., Ltd, Shanghai, China) according to the manufacturer's instructions.
Ecl chemiluminescence detection kit
Manufactured by Sangon
Sourced in China
The ECL chemiluminescence detection kit is a laboratory equipment designed for the detection and quantification of proteins in Western blot analysis. It utilizes a chemiluminescent substrate to produce a luminescent signal that can be captured and measured using a compatible imaging system.
Automatically generated - may contain errors
2 protocols using ecl chemiluminescence detection kit
1
Polyclonal Antibody Detection in Chicken
2
Western Blot Analysis of p53 Expression
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Osteoblasts were lysed with 1 ml RIPA buffer (Guangzhou RiboBio Co., Ltd.) to extract total protein. Protein samples were quantified using a bicinchoninic acid kit (Sangon Biotech Co., Ltd.), followed by denaturation in boiling water for 5 min. Electrophoresis was performed using 12% SDS-PAGE to separate proteins (30 µg per well) according to their molecular weights. Proteins were transferred to a PVDF membrane and blocking was carried out in 5% non-fat milk for 2 h at room temperature. Primary antibodies of rabbit GAPDH (1:1,200; cat. no. ab181602; Abcam) and p53 (1:1,200; cat. no. ab131442; Abcam) were used to incubate the membranes for 15 h at 4˚C. Horseradish peroxidase goat anti-rabbit (immunoglobulin G; 1:1,100; cat. no. ab6721; Abcam) secondary antibody was then used to blot the membranes further at room temperature for 2 h. The ECL Chemiluminescence Detection kit (Sangon Biotech Co., Ltd.) was used to develop protein signals. Gray values were normalized using ImageJ version 1.46 (National Institutes of Health).
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