The RAW264.7 macrophage cell line was purchased from the Institute of Biochemistry and Cell Biology (Shanghai Institute for Biological Science, the Chinese Academy of Sciences, Shanghai, China). DMEM and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT, USA). RhTrx was purchased from R&D Systems (Minneapolis, MN). Ox-LDL, oil red O, hematoxylin, DMSO and a protease inhibitor cocktail were purchased from Sigma (St. Louis, MO, USA). FITC Annexin V Apoptosis Detection Kit I was purchased from Becton Dickinson (Franklin Lakes, NJ, USA). SB203580 and anisomycin were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit monoclonal antibodies against p38 MAPK, p-p38 MAPK, Bcl-2, Bax, and cleaved caspase-3 and goat anti-rabbit IgG HRP-conjugated antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit monoclonal antibody against LOX-1 and goat anti-rabbit IgG (H+L) TRITC-conjugated antibody were purchased from Abcam (Cambridge, MA, USA). A ROS Assay Kit was purchased from Beyotime Biotech (Shanghai, China).
Hrp conjugated goat anti rabbit igg antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The HRP-conjugated goat anti-rabbit IgG antibody is a secondary antibody that binds to rabbit primary antibodies. The horseradish peroxidase (HRP) enzyme conjugated to the antibody allows for detection and visualization of the target protein in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Mouse anti β actin antibody, by Cell Signaling Technology (2 mentions)
Ecl western blotting detection reagent, by GE Healthcare (2 mentions)
Protein g plus agarose bead, by Santa Cruz Biotechnology (2 mentions)
Normal mouse igg antibody, by Santa Cruz Biotechnology (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Hrp conjugated horse anti mouse igg antibody, by Cell Signaling Technology (2 mentions)
Anti kif5, by Merck Group (2 mentions)
Hrp conjugated goat anti mouse igg light chain specific antibody, by Jackson ImmunoResearch (2 mentions)
Protein g plus agarose beads, by Thermo Fisher Scientific (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Anisomycin, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Ox ldl, by Merck Group (1 mentions)
Sb203580, by Cell Signaling Technology (1 mentions)
Any kd tris glycine gels, by Bio-Rad (1 mentions)
Sds page 10 gel, by Bio-Rad (1 mentions)
22 protocols using hrp conjugated goat anti rabbit igg antibody
1
Oxidized LDL-induced Macrophage Apoptosis
2
Quantifying Urinary AQP1 Levels
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The precipitated proteins were mixed with β-mercaptoethanol, incubated in a boiling water bath for five minutes, and loaded onto precast Any-kD Tris-glycine gels (Bio-Rad Laboratories Inc., Hercules, CA, USA) for electrophoresis so that each well contained 60 μg of creatinine per sample. Normalization to creatinine with urine stored in these conditions is the standard for urinary protein processing via western blotting [16 (link)]. Proteins were transferred onto PVDF membranes and blocked with 5% nonfat milk in PBS-Tween 20. Blocked membranes were incubated overnight with anti-AQP1 (H-55) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), diluted 1 : 500 in blocking buffer. After washing, the membranes were incubated with a 1 : 2000 dilution of goat anti-rabbit IgG HRP-conjugated antibody (Cell Signaling Technology, Danvers, MA, USA) for two hours at room temperature and visualized by chemiluminescence. AQP1 levels were semiquantified in arbitrary units using ImageJ software for area under the curve (AUC) analysis.
3
Western Blot Analysis of Autophagy and Inflammation Markers
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Total protein was extracted from tissues and NSCLC cells using ProteoPrep Sample Extraction kit (Sigma-Aldrich; Merck KGaA). The QuantiPro™ BCA Assay kit (cat. no. QPBCA; Sigma-Aldrich; Merck KGaA) was used to measure protein concentration. Protein samples (30 µg) were separated via SDS-PAGE (10% gel; Bio-Rad Laboratories, Inc.) and transferred to PVDF membranes. The membranes were then blocked with 5% non-fat milk in TBS with 0.05% Tween-20 for 1 h at room temperature. After the membranes were incubated with primary antibodies at 4˚C overnight, secondary antibodies were added and incubated with the membranes at room temperature for 1 h. The bands were visualized with an ECL Western Blotting substrate kit (cat. no. K820; BioVision, Inc.) and the protein expression was quantified using ImageJ v1.8.0 software (National Institutes of Health) with GAPDH as the loading control. The primary antibodies used included: Mouse anti-IL-1β (1:1,000; cat. no. 12242; Cell Signaling Technology, Inc.), rabbit anti-LC3B (1:1,000; cat. no. ab51520; Abcam), rabbit anti-P62 (1:10,000; cat. no. ab109012; Abcam) and rabbit anti-GAPDH (1:2,500; cat. no. ab9485; Abcam). The corresponding secondary antibodies included: Anti-mouse IgG HRP-conjugated antibody (1;2,000; cat. no. 7076; Cell Signaling Technology, Inc.) and goat anti-rabbit IgG HRP-conjugated antibody (1:20,000; cat. no. ab205718; Abcam).
4
Immunoblotting Analysis of APOBEC3A Expression
5
Formononetin Modulates HUVEC Angiogenesis
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Human Umbilical Vein Endothelial Cells (HUVEC) were obtained from Thermo Fisher Scientific Inc. (NYSE: TMO), maintained in Vascular Cell Basal Medium ATCC (Manassas, VA), and used prior to passage 7. Fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin-streptomycin (PS) and 0.25% (w/v) trypsin/1 mM EDTA were all purchased from Invitrogen (Carlsbad, CA, USA). Endothelial cell growth supplement (ECGS), heparin and gelatin were all supplied by Sigma (St Louis, MO). Vascular endothelial growth factors (VEGF) were obtained from R&D Systems (Minneapolis, MN). Anti-MMP2/9 antibody, Anti-ROCK-II antibody, anti-MYPT1 antibody, Anti-LIMK1 antibody, Anti-cofilin antibody, Anti-MLC2 antibody, Anti-β-actin antibody and goat anti-rabbit IgG HRP-conjugated antibody were all purchased from Cell Signaling Technology (Berverly, MA). Anti-ERα Antibody was purchased from Santa Cruz Biotechnology, Inc. Dimethyl sulfoxide (DMSO) was acquired from SIGMA. Formononetin was obtained from the Chinese National Institute for Control of Pharmaceutical and Biological Products (Beijing, China) and was dissolved in DMSO to form a 50 mM solution.
6
Western Blot Analysis of Fgf21 in Liver
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Liver tissue was lyzed in ice-cold lysis buffer (Tissue Protein Extraction Reagent; Thermo Fisher Scientific) supplemented with complete protease and phosphatase inhibitor cocktail and EDTA (Thermo Fisher Scientific). After centrifugation at 10,000 g and 4 °C for 5 min, proteins were boiled in Roti-Load1 Buffer (Carl Roth, Karlsruhe, Germany). Proteins (30 μg) were separated on SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were incubated overnight at 4 °C with rabbit anti-Fgf21 antibody (diluted 1:1,000; Abcam, Cambridge, UK) and rabbit anti-Gapdh antibody (diluted 1:5,000; Cell Signaling, Frankfurt, Germany) and then with secondary goat anti-rabbit IgG, HRP-conjugated antibody (1:5,000; Cell Signaling) for 1 h at room temperature. Antibody binding was detected with ECL detection reagent, and densitometric analysis was performed using Image Lab software 6.1 (Bio-Rad Laboratories). Data are shown as ratio of Fgf21 protein over loading control Gapdh, normalized to wild type mice (etb+/+).
7
Quantitative Analysis of Apoptosis Markers
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Total proteins from B cells and T cells were extracted (Beyotime, China) and quantified using the BCA kit (Pierce, Rockford, IL). Lysates were separated on 12 % SDS–polyacrylamide gel electrophoresis and transferred to PVDF (Millipore, USA), followed by blocking in TBS/0.1 % Tween 20 with 5 % nonfat dry milk. Antibodies were used as following: rabbit anti-mouse Caspase 3 antibody (1:1000); goat anti-rabbit IgG HRP-conjugated antibody (1:2000); and mouse anti-β-actin antibody (1:1000) (Cell Signaling Technology).
8
Immunoblot Analysis of Legionella Virulence Factors
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L. pneumophila strains were grown in AYE broth at 37°C to either mid-log or late-log phase, at which times the OD660 was measured and the cultures were centrifuged. The resulting supernatants were filtered through a 0.2 μm syringe filter. The supernatant and pellets were diluted in PBS and 2X Laemmli buffer according to the OD660 to normalize for bacterial numbers. The samples were subjected to SDS-PAGE and then analyzed by immunoblot as previously described52 (link). Briefly, after blocking in 5% milk for 1 h, the blots were incubated overnight with 1:5,000 dilutions of rabbit anti-CelA, anti-ProA, anti-ICDH or 1:1,000 dilutions of anti-LegP antibodies, washed, and then incubated for 1 h with 1:1,000 dilution of secondary HRP-conjugated goat anti-rabbit IgG antibody (Cell Signaling Technology). Images of the immunoblots were developed with ECL™ Western Blotting Detection Reagent (GE Healthcare).
9
Western Blot Analysis of FcRY Expression
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The expression pattern of FcRY was examined using western blotting analysis. Each tissue sample from the chickens [white ovarian follicle, theca layer, and granulosa cell (GC) layer of yellow ovarian follicle] was homogenized with lysis buffer. The egg yolk and tissue homogenate samples (equivalent to 2 or 5 µg of protein) were separated using SDS-PAGE under reducing conditions. The blotting PVDF membranes were first incubated with rabbit anti-chicken FcRY antibody (1:2,500) or rabbit anti-human GAPDH (1:1,000; Santa Cruz Biotechnology, Dallas, TX) and then with HRP-conjugated goat anti-rabbit IgG antibody (1:2,000; Cell Signaling technology, Danvers, MA). The membranes were visualized using a chemiluminescence detection method (SuperSignal® West Dura Extended Duration Substrate; Thermo Fisher Scientific) with a charge-coupled device camera (AE-6960/C; Atto, Tokyo, Japan).
10
Immunoprecipitation of Synaptic Proteins
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