Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting were performed as described previously [29 (link)]. Cell lysates were prepared in lysis buffer [10 mM HEPES-KOH, pH 7.4, 100 mM KCl, 1 mM MgCl2, 1% Triton X-100, and protease inhibitor cocktails (Nacalai)]. After incubation for 10 min on ice, the lysate was clarified by centrifugation at 14,000 × g for 10 min. The supernatants were then subjected to SDS–PAGE on a 7.5% gel (Bio-Rad). The gel was electrotransferred to polyvinylidene fluoride or polyvinylidene difluoride membranes (pore size: 0.45 μm, Merck Group Japan, Tokyo, Japan). The proteins on the membrane were detected by incubation with diluted anti-AhR, anti-ARNT (Santa Cruz Biotechnology, Cosmo Bio Co., LTD, Tokyo, Japan), and anti-γ-tubulin (Sigma, Sigma-Aldrich Japan, Tokyo, Japan) followed by HRP-conjugated anti-mouse immunoglobulin G (Cell Signaling, CST Japan, Tokyo, Japan) using a charge-coupled device camera.
Hrp conjugated anti mouse immunoglobulin g
Manufactured by Cell Signaling Technology
Sourced in Japan
HRP-conjugated anti-mouse immunoglobulin G is a laboratory reagent used in various immunoassay techniques. It consists of horseradish peroxidase (HRP) enzyme covalently coupled to antibodies that specifically recognize mouse immunoglobulin G (IgG) molecules. This reagent can be used to detect and quantify the presence of mouse IgG in biological samples.
Automatically generated - may contain errors
2 protocols using hrp conjugated anti mouse immunoglobulin g
1
SDS-PAGE and Western Blotting for Protein Analysis
2
Evaluating BbZIP Protein Expression in E.coli
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E.coli cells (C43 strain) transformed with the empty vector or the plasmids harboring the genes encoding BbZIP or the variants were allowed to grow in the autoinduction media overnight before they were applied on LB agar plates containing indicated concentrations of ZnCl2. After 24 hours of incubation at 37°C, the plates were imaged. To estimate expression of BbZIP and the variants, cell lysates were mixed with SDS sample loading buffer, applied to SDS-PAGE, and transferred to a PVDF membrane. After being blocked with 5% nonfat dry milk, the membrane was incubated with a mouse custom anti-BbZIP monoclonal antibody at 0.06 µg/ml at 4°C overnight (32 (link)). The bound primary antibodies were detected with an HRP-conjugated anti-mouse immunoglobulin-G at 1:5000 dilution (Cell Signaling Technology, Catalog# 7076S) by chemiluminescence (VWR). The images of the blots and plates were taken using a Bio-Rad ChemiDoc Imaging System.
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