Proteins of lung were extracted with using protein extraction reagents (Thermo Fisher Scientific Inc., USA) and the concentration was tested using BCA protein assay (Sigma-Aldrich, USA). Proteins (30 μg) were separated by SDS–polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (BioRad, Hercules, CA, USA). Membranes were blocked and then incubated with the following primary antibodies: anti-NF-kBp65 (ab16502), anti-NF-kBp65 (phospho S536) antibody (ab86299), anti-superoxide dismutase 1 antibody [SOD1] (ab20926), anti-SOD2/MnSOD antibody (ab13533), anti-Catalase antibody (ab16731), and anti-beta Actin antibody (ab8227). After primary antibody incubation, membranes were washed, incubated with alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG antibodies (Promega, Madison, WI, USA), and quantified and digitally analyzed using the image J program (NIH).
Alkaline phosphatase conjugated anti mouse or anti rabbit igg antibodies
Manufactured by Promega
Sourced in United States
Alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG antibodies are secondary antibodies that are conjugated with the enzyme alkaline phosphatase. These antibodies are used to detect and quantify the presence of mouse or rabbit primary antibodies in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry.
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4 protocols using alkaline phosphatase conjugated anti mouse or anti rabbit igg antibodies
1
Protein Expression Analysis of Lung Tissues
2
Lung Protein Analysis via Western Blot
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Proteins of lung were extracted with using protein extraction reagents (Thermo Fisher Scientific Inc., USA) and the concentration was tested using BCA protein assay (Sigma-Aldrich, USA). Proteins (30 μg) were separated by SDS–polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (BioRad, Hercules, CA, USA). Membranes were blocked and then incubated with the following primary antibodies: Anti-Keap1 antibody [1F10B6] (ab150654), Anti-Nrf2 antibody (ab31163), Anti-pan-AKT antibody (ab8805), Anti-pan-AKT (phospho T308) antibody (ab38449), anti-Catalase antibody (ab16731), and anti-beta Actin antibody (ab8227). After primary antibody incubation, membranes were washed, incubated with alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG antibodies (Promega, Madison, WI, USA), and quantified and digitally analyzed using the image J program (NIH).
3
Immunoblotting Analysis of Tight Junction Proteins
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Proteins were extracted with protein extraction reagents (Thermo Fisher Scientific Inc., USA). Proteins (30 µg) were separated by SDS–polyacrylamide gel electrophoresis and electrophoretically transferred to apolyvinylidene difluoride (PVDF) membrane (BioRad, Hercules, CA, USA). Membranes were blocked and then incubated with the following primary antibodies: ZO-1 (ab59720), Claudin1 (ab115225), and Occludin (ab31721) (Abcam, Inc., USA). Mouse β-actin antibody (Sigma) was used for protein loading control. After primary antibody incubation, membranes were washed, incubated with alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG antibodies (Promega, Madison, WI, USA), and quantified and digitally analyzed using the image J program (NIH).
4
Protein Expression Analysis of Intestinal Tissues
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One piece of jejunum, ileum, and ileum were harvested and stored at –80°C. Proteins were extracted with using protein extraction reagents (Thermo Fisher Scientific Inc., USA) and the concentration was tested using BCA protein assay (Sigma-Aldrich, USA). Proteins (30 μg) were separated by SDS–polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (BioRad, Hercules, CA, USA). Membranes were blocked and then incubated with the following primary antibodies: Anti-CCR7 antibody [Y59] (ab32527) (Abcam). Mouse β–actin antibody (Sigma) was used for protein loading control. After primary antibody incubation, membranes were washed, incubated with alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG antibodies (Promega, Madison, WI, USA), and quantified and digitally analyzed using the image J program (NIH).
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