Rat kidney microsomes (10 µg per lane) were incubated at 100 °C for 5 min in Laemmli sample buffer (BioRad, Hercules, CA, USA) and electrophoresed 1 h constant voltage (150 V) through precast polyacrylamide gels (BioRad). Microsomal proteins were then transferred electrophoretically (30 min at 20 V) to PVFD membrane and incubated with CYP4A1/CYP4A2/CYP4A3 rabbit anti-rat polyclonal antibody at a 1:1000 dilution (Lifespan Biosciences) overnight at 4 °C. After washes with PBST, the secondary antibody (goat anti-rabbit conjugated to horseradish peroxidase, Thermo Scientific, Waltham, MA, USA) was added at a 1:5000 dilution for 1 h at room temperature. The immunoreactive proteins were detected via enhanced chemiluminescence and X-ray film imaging, and the resultant signals were analyzed by densitometry (Bio-Rad). The intensity of the band was normalized to GADPH signal, which was used as loading control.
Goat anti rabbit conjugated to horseradish peroxidase
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Goat anti-rabbit conjugated to horseradish peroxidase is a secondary antibody used in immunoassays, such as Western blotting and ELISA, to detect the presence of rabbit primary antibodies. The horseradish peroxidase (HRP) enzyme conjugated to the goat anti-rabbit antibody serves as a reporter molecule, allowing for the visualization and quantification of the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Laemmli sample buffer, by Bio-Rad (2 mentions)
Densitometry, by Bio-Rad (2 mentions)
Precast polyacrylamide gel, by Bio-Rad (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Rabbit anti mch, by Abcam (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Rabbit anti hla igg antibody, by Merck Group (1 mentions)
Las 4000, by Cytiva (1 mentions)
4 protocols using goat anti rabbit conjugated to horseradish peroxidase
1
Quantitative Western Blot Analysis of Rat Kidney CYP4A Isoforms
2
Western Blot Analysis of Fluorescent Protein Constructs
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Western blots from mCh, RH1-mCh and RH2-mCh expressed in tsa201 (HEK cells stably transformed with SV40) cells were performed according to published methods [22 (link)]. Briefly cell homogenates were run on a 10% SDS-PAGE, transferred to a PVDF membrane and incubated with the primary antibody rabbit anti-mCh (AbCam, England) and the secondary antibody goat anti-rabbit conjugated to horseradish peroxidase (Novex, ThermoFisher). Membranes were developed with the SuperSignal West Dura Extended Duration Substrate (ThermoFisher Scientific).
3
Detecting Hla Protein by Western Blot
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To detect the production of Hla, stationary-phase culture supernatant was collected and heated for 10 min at 95°C. The samples were then separated by 12% SDS-PAGE and electrotransferred onto a polyvinylidene difluoride membrane (GE, Piscataway, NJ). After blocking with 5% (w/v) nonfat milk in TBST buffer at room temperature for 1 h, the membrane was incubated with a rabbit anti-Hla IgG antibody (Sigma) at a 1/2500 dilution. Bound antibody was detected with the goat anti-rabbit conjugated to horseradish peroxidase (ThermoFisher) at a 1/5000 dilution. The images were obtained with ImageQuant LAS 4000 (GE Healthcare).
4
Western Blot Quantification of Rat Kidney CYP4A
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Rat kidney microsomes (10 ug per lane,) were incubated at 100°C for 5 minutes in Laemmli sample buffer (BioRad, Hercules, CA) and electrophoresed 1 hour constant voltage (150 V) through precast polyacrylamide gels (BioRad) with minor modifications [33 (link)]. Microsomal proteins were then transferred electrophoretically (30 min at 20 V) to PVFD membrane and incubated with CYP4A1/CYP4A2/CYP4A3 rabbit anti-rat polyclonal antibody, at a 1 : 1000 dilution (Lifespan Biosciences) overnight at 4°C. After washes with PBST, the secondary antibody (goat anti-rabbit conjugated to horseradish peroxidase, Thermo Scientific) was added at a 1 : 5000 dilution for 1 hour at room temperature. The immunoreactive proteins were detected via enhanced chemiluminescence and X-ray film imaging and the resultant signals were analyzed by densitometry (BioRad). The intensity of the band was normalized to GADPH signals, which was used as loading control.
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