Ten-to-twelve 10-day-old seedlings were induced for 24 h with estradiol, flash frozen, then ground in 500 µl freshly prepared lysis buffer (25 mM Tris HCl pH 8, 150 mM NaCl, 1% SDS, 1 × cOmplete ULTRA protease inhibitor (Roche), and 1 × PhosSTOP (Roche)). Lysates were cleared via 14,000 × g centrifugation at 4 °C, and split evenly for RNA vs protein processing. RNA extraction, cDNA synthesis, and RT-qPCR were carried out as described above. Lysates for protein work were mixed 1:1 with 2 × Laemmli sample buffer, and boiled for 1 min. Proteins were resolved via SDS-PAGE, blotted to Hybond ECL membrane (GE Healthcare), and blocked in 5% milk fat. PHB variants were detected via anti-GFP primary antibodies (600-406-215; Rockland Immunochemicals; 1:500 dilution) and anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch; 1:5,000 dilution). Detection of secondary antibodies was performed with SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific). Blots were scanned and quantified using ImageJ. Four independent biological replicates were performed, each with two technical replicates.
Anti rabbit horseradish peroxidase conjugated secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States, Cameroon
Anti-rabbit horseradish peroxidase-conjugated secondary antibody is a laboratory reagent used for detection and quantification of rabbit primary antibodies in various immunoassay techniques. The antibody is conjugated to the enzyme horseradish peroxidase, which catalyzes a chromogenic or chemiluminescent reaction, allowing the visualization and measurement of the target rabbit antibody.
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Lab products found in correlation
Speedy protocol, by Eurogentec (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Enhanced chemiluminescence, by GE Healthcare (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Phospho s6 ribosomal protein, by Cell Signaling Technology (2 mentions)
Rabbit anti cx26 antibody, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated anti mouse secondary antibody, by GE Healthcare (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Hybond ecl membrane, by GE Healthcare (1 mentions)
Complete ultra protease inhibitor, by Roche (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Phosstop, by Roche (1 mentions)
Rabbit anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Ecl plus kit, by Cytiva (1 mentions)
Storm 860 scanner, by GE Healthcare (1 mentions)
Immobilon kit, by Merck Group (1 mentions)
Acrylamide gel, by Bio-Rad (1 mentions)
Image lab software v 6, by Bio-Rad (1 mentions)
Ecl plus western blotting detection system, by GE Healthcare (1 mentions)
29 protocols using anti rabbit horseradish peroxidase conjugated secondary antibody
1
Estradiol-Induced Protein Profiling
2
Quantification of Hippocampal β-Catenin
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Hippocampal proteins (∼20 µg, 4–5 mice/group) were extracted and assayed (BioRad), separated using a 10% SDS PAGE and transferred to nitrocellulose membranes for the detection of β-catenin protein levels (rabbit anti-β-catenin, Santa Cruz, California, USA). Membranes were subsequently incubated (1 h) with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:2000; Jackson ImmunoResearch, West Grove, PA, USA) in TBST blocking buffer (50 mM Tris-HCl, pH = 7.5; 150 mM NaCl; 0.1% Tween 20) containing 5% skim milk, and visualized with enhanced chemiluminescence (ECL Plus kit; Amersham, Baie d’Urfé, QC, Canada) using a phosphorImager (Scanner STORM 860; GE Healthcare, Baie d’Urfé, QC, Canada). Band intensity was quantified by densitometry with Scion Image (Molecular Dynamics, Sunnyvale, CA, USA).
3
Antibody Production and Detection of LppM
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Recombinant 6His‐LppM26–227 purified from E. coli was used to generate specific rabbit antibodies against LppM according to the Speedy protocol (Eurogentec). Proteins were resolved by SDS‐PAGE using 4–15% acrylamide gels (BioRad) and then transferred onto a polyvinylidene difluoride membrane. The membrane was saturated with TBS‐Tween‐5% milk and probed overnight with anti‐LppM antibodies diluted 1:1000 with TBS‐Tween‐3% milk. The membrane was then incubated with anti‐rabbit horseradish peroxidase‐conjugated secondary antibodies (Jackson Immunoresearch) for 1 h at room temperature, followed by detection using Immobilon kit (Millipore). Chemiluminescence was determined using the ImageQuant LAS 4000 (GE Healthcare).
4
Western Blot Analysis of Breast Cancer Cells
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MBCDF, MBCD17, MBCD3 and MBCDF-D5 breast cancer cells were lysed in a buffer containing 50 mM HEPES (pH 7.4), 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 10 mM NaF, 50 mM β-glycerophosphate, 1 mM Na3VO4 and 1X protease inhibitor cocktail (Complete EDTA-free; Roche Diagnostics). Protein was quantified using a Bradford assay (Bio-Rad Laboratories, Inc.). Whole protein extract (20 µg) was separated by 9 and 12% SDS-PAGE then transferred to Immobilon-P PVDF membranes (MilliporeSigma). The membranes were blocked with 5% skim milk in PBS-Tween (0.1%) for 1 h at room temperature. After this time, primary antibodies were added and incubated overnight at 4°C. Membranes were washed and then incubated with anti-mouse horseradish peroxidase--conjugated secondary antibodies (cat. no. 115-035-003; 1:10,000; Jackson ImmunoResearch Laboratories, Inc.) or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (cat. no. 111-035-003; 1:10,000; Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. Proteins were visualized using the ECL plus western blotting detection system (GE Healthcare) and blot images were digitized using Chemidoc (Bio-Rad Laboratories, Inc.). Densitometry of the bands was measured using ImageLab software v6 (Bio-Rad Laboratories, Inc.).
5
Immunohistochemical Staining of Paraffin-Embedded Tissue
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Tissues were fixed in 4% formaldehyde or paraformaldehyde overnight, embedded in paraffin and sliced in 4 µm sections. Antigen retrieval was performed at 100 °C in a citrate buffer, pH 6 for 20 min. Sections were blocked in 2.5% BSA in PBS with 0.1% Tween20 and RFP (AB223), or Ki67 (SP6) was used as the primary antibody. Anti-rabbit horseradish peroxidase-conjugated secondary antibody was used for development (Jackson ImmunoResearch). Counterstaining was performed with DAPI (Vector Laboratories, Burlingame, CA, USA).
6
Evaluating Claudin-5 Expression in Mouse Brain
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Left brain hemispheres from mice in the 99mTc-albumin experiments were immediately flash frozen in liquid nitrogen and stored at −80 °C for ten 99mTc-albumin half-lives (~60 h). Hippocampus and striatum were dissected and sonicated in radioimmunoprecipitation assay (RIPA) buffer with protease/phosphatase inhibitors (Thermo; Rockford, IL 1:100). Samples were then centrifuged for 15 min at 12000 × g at 4 °C and supernatant collected and standardized with a bicinchoninic acid (BCA) assay. Brain extracts of equivalent total protein content were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (20 µg total protein/lane) under reducing conditions, transferred to nitrocellulose, and probed with rabbit polyclonal claudin-5 (Invitrogen, Eugene, OR; 1:1000) and rabbit monoclonal β-actin (Cell signaling, Danvers, MA; 1:10000) as loading controls. Blots were then probed with anti-rabbit horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch; 1:5000) and visualized by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ). Optical densities of the immunoreactive protein bands were quantified using Image-Quant with background subtraction and normalization to loading control.
7
Quantitative Western Blot Analysis
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Organotypic cultures were scraped from tissue culture plates and homogenized in RIPA lysis buffer that contained protease and phosphatase inhibitors (Thermo Scientific). Protein content of culture lysates was measured using Micro BCA protein assay kit (Thermo Scientific). Proteins were separated via electrophoresis in 12% Tris-Glycine Mini Gels (Life Technologies), transferred to a PVDF membrane, and stained with antibodies (1:10,000 rabbit antibodies to phospho-Akt (Ser473) (D9E), phospho-Akt (Thr308) (C31E5E), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E), phospho-S6 (Ser235/236) (D57.2.2E), phospho-S6 (Ser 240/244) (D68F8), phospho−IGF-1Rβ (Tyr1135/1136)/Insulin Receptor β (Tyr1150/1151) (19H7, used at 1:500 concentration), total Akt (C67E7), p44/42 MAPK (Erk1/2) (137F5), S6 Ribosomal Protein (5G10), and IGF-1Rβ (D23H3, used at 1:1000 concentration), all from Cell Signaling Technology, and anti-rabbit horseradish peroxidase-conjugated secondary antibody from Jackson ImmunoResearch Laboratories). Bands were visualized using Pierce enhanced chemiluminescence (ECL) substrate on CL-XPosure X-ray films (Thermo Scientific), scanned, and quantified using densitometry via ImageJ software (NIH).
8
Quantifying Tight Junction Protein ZO-1 by Western Blot
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Individual brain extracts of equivalent total protein content were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (50 μg total protein/lane) under reducing conditions, transferred to nitrocellulose, and probed with 1 μg/ml of rabbit polyclonal antibodies to ZO-1 (Invitrogen). Blots were then probed with anti-rabbit horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) (1 μg/ml) and visualized by enhanced chemiluminescence (GE Healthcare LifeSciences, Piscataway, NJ). Densities of the bands were quantified using ImageJ.
9
Immunohistochemical Staining of Chimeric Mouse Livers
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Cryostat sections of chimeric mouse livers were stained as previously described (Lutgehetmann et al., 2012 (link)). Briefly, sections were fixed with acetone and incubated with mouse anti-CK18 (1:400, Dako, Glostrup, Denmark), rabbit anti-HBcAg (1:2000, Dako), mouse HLA-ABC (1:50, Antibodies-online, Aachen, Germany), and human anti-Delta (anti-HDAg-positive human serum, 1:8,000). Specific signals were visualized with Alexa 488-, 555-, or 633-labeled secondary antibodies (Invitrogen, Darmstadt, Germany). To enhance the HBcAg staining an anti-rabbit horseradish-peroxidase conjugated secondary antibody (Jackson Immunoresearch, Suffolk, United Kingdom) and the TSA Fluorescein System (Perkin Elmer, Jügesheim, Germany) were used. Nuclear staining was achieved by Hoechst 33258 (1:20,000 diluted, Invitrogen, Waltham, MA, United States). Stained sections were then mounted with fluorescent mounting media (Dako) and analyzed with the fluorescence microscope BZ8710 (Keyence, Osaka, Japan) using the same settings for the different experimental groups. The percentages of HDAg-positive human hepatocytes were estimated as previously described (Lutgehetmann et al., 2012 (link)) and by using 2–5 visual fields (displaying an average of 500 human hepatocytes) per mouse liver.
10
Mitochondrial Protein Profiling by Western Blot
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Western blot analysis was conducted on isolated SS mitochondria to determine the protein content of nSMase2 and nSMase3. Isolated SS mitochondria were diluted to 1µg/µL in Laemmeli buffer (Laemmli, 1970) and electrophoretically separated using 7.5% standard glycine-based SDS-PAGE. Separated proteins were transferred to a polyvinylidene fluoride membrane (Bio-Rad, Mississauga, ON) using a semi-dry Trans-Blot Turbo system (Bio-Rad). Membranes were immunoprobed with primary antibodies directed against nSMase2 and nSMase3 (ECM Biosciences, Versailles, KY) diluted in 5% (w/v) milk in tris-buffered saline then anti-rabbit horseradish peroxidase conjugated secondary antibody (Jackson Immuno Research Labs, West Grove, PA), and visualized with Clarity Western ECL substrate (Bio-Rad Inc) and the Li-Cor imaging system (LI-COR Biosciences, Lincoln, NE). Ponceau stain (Bioshop, Burlington, ON) was used to normalize protein loading.
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