CD8+ T cells (5×106) were lysed with RIPA buffer containing HaltTM protease and phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL) on ice for 30 minutes4 (link), 27 (link). Samples were run by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked using buffer containing 2% BSA and 0.5% sodium azide in TBST for 1 hour and incubated with rabbit polyclonal CYP11A1 antibody (Lifespan Biosciences, Seattle, WA) overnight at 4°C. Horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare, Hertfordshire, UK) was used to detect CYP11A1 protein. Mouse monoclonal anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO) was used as an internal control. Immunoreactive bands were quantified by densitometric quantification of autoradiographs using Image J (NIMH, Bethesda, MD), and expressed as relative CYP11A1 normalized to β-actin (Sigma-Aldrich, St. Louis, MO).
Horseradish peroxidase conjugated anti rabbit igg
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
Horseradish peroxidase-conjugated anti-rabbit IgG is a secondary antibody used for immunodetection. It consists of an anti-rabbit IgG antibody covalently linked to the enzyme horseradish peroxidase. This conjugate can be used to detect and quantify rabbit primary antibodies in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated anti mouse igg, by GE Healthcare (4 mentions)
β actin, by Merck Group (3 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
Anti β actin antibody, by Merck Group (2 mentions)
Cyp11a1 antibody, by LifeSpan BioSciences (2 mentions)
Ecl plus western blotting detection system, by GE Healthcare (2 mentions)
Ecl western blotting reagent, by GE Healthcare (2 mentions)
Las 3000 luminescent image analyzer, by Fujifilm (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
Erk5 antibody, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Anti actin, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti rat igg, by Santa Cruz Biotechnology (1 mentions)
Anti uhrf1, by Santa Cruz Biotechnology (1 mentions)
Anti flag, by Merck Group (1 mentions)
Chemi lumi one l, by Nacalai Tesque (1 mentions)
Hybond p, by GE Healthcare (1 mentions)
Las 1000 uv mini, by Fujifilm (1 mentions)
Immobilon western chemiluminescent substrate, by Merck Group (1 mentions)
32 protocols using horseradish peroxidase conjugated anti rabbit igg
1
Quantification of CYP11A1 Protein Levels
2
Western Blot Analysis of Protein Expression and ERK5 Phosphorylation
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Western blotting analysis for protein expression and ERK5 phosphorylation in mice aortas or HUVECs was performed as described previously [6 (link)]. Briefly, lysates of aortas or HUVECs were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electro-transferred to polyvinylidene difluoride membranes, as described previously. The membranes were blocked in phosphate-buffered saline with 0.1% Tween-20 (PBS-T), containing 4% milk powder for 90 min at room temperature and then incubated overnight at 4 °C with one of the following primary antibodies: VCAM-1 antibody (sc-8304; Santa Cruz, Santa Cruz, CA, USA), phospho-ERK5 antibody (#3371; Cell Signaling, Danvers, MA, USA), ERK5 antibody (#3372; Cell Signaling), or β-actin antibody (as a loading control, #3700; Cell Signaling). The membranes were subsequently incubated with horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare Biosciences, Piscataway, NJ, USA) for 1 h at room temperature, and the signals were developed using ECL Plus Western Blotting Detection System (GE Healthcare Biosciences, Piscataway, NJ, USA). Immunoreactive bands were quantified by densitometry in the linear range of film exposure using a UMAX Astra 2200 scanner and image J v. 1.37 software (National Institutes of Health, Bethesda, MD, USA).
3
Western Blot Quantification of Polyamine Modulon Proteins
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Western blot analysis was performed by the method described in [47 (link)] using horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare Bio-Sciences) as a secondary antibody and ECL Western blotting reagents (GE Healthcare Bio-Sciences). Antibodies against the proteins encoded by polyamine modulon were prepared as described previously [21 (link),24 (link),26 (link),27 (link),48 (link)]. The level of protein on the blot was quantified with a LAS-3000 luminescent image analyzer (Fuji Film).
4
Western Blot Analysis of Brain Development
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Western blot analysis was performed as described previously.55 (link) In brief, isolated E11, E14, E18, P7 and adult brains were lysed in lysis buffer (1% Nonidet P-40, 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 100 μM protease inhibitor cocktail (Nacalai Tesque, 03969), 1 mM EDTA). The protein samples were separated in gradient (5–20%) polyacrylamide gels (e-PAGEL; ATTO, 2331830), transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, RPN303F), and probed with anti-FLAG (1:2000; Sigma, F1804), anti-Np95 (1:2000),32 (link) anti-UHRF1 (1:2000; Santa Cruz Biotechnology, sc-98817), anti-DNMT1 (1:2000; Cosmo Bio, BAM-70-201-EX) or anti-actin (1:2000; Abcam, ab3280) antibody. Horseradish peroxidase-conjugated anti-mouse IgG (1:5000; GE Healthcare Life Sciences, NA931), horseradish peroxidase-conjugated anti-rabbit IgG (1:5000; GE Healthcare Life Sciences, NA934) or horseradish peroxidase-conjugated anti-rat IgG ( 1:5000; Santa Cruz Biotechnology, sc-2006) was used as the secondary antibody. Detection was performed using Chemi-Lumi One L (Nacalai Tesque, 07880).
5
Western Blot Analysis of AS3MT Protein
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HepG2 cells treated with each siRNA were washed with ice-cold PBS(−) and collected with 50 mM Tris–HCl (pH 7.4). The pellets were homogenized by sonication for 15 min, and then centrifuged at 105,000 × g for 1 h. A 20 μg portion of protein in the supernatant was separated by SDS-PAGE, and then transferred onto polyvinylidene fluoride membrane (Hybond-P, GE Healthcare) at 20 V for 1 h. The membrane was blocked for 1 h with 3% BSA in PBS(−) containing 0.1% Tween-20 (PBS-T). For the detection of AS3MT, the membrane was washed briefly with PBS-T and incubated with anti-Cyt19 (AS3MT) rabbit polyclonal antibody (1:1000) (Santa Cruz Biotechnology, Dallas, TX, USA) diluted 100-fold with PBS-T containing 5% BSA overnight at 4 °C. The membrane was washed with PBS-T, and probed with horseradish-peroxidase-conjugated anti-rabbit IgG (1:50,000) (GE Healthcare). The bands were visualized with Immobilon Western Chemiluminescent Substrate (Merck Millipore, Billerica, MA, USA) and LAS-1000 UV mini (FUJIFILM, Tokyo, Japan). For the detection of rhAS3MT, anti-His-tag antibody (GE Healthcare) was also used as the primary antibody in addition to anti-Cyt19 antibody. Anti-GAPDH mouse polyclonal antibody (Santa Cruz Biotechnology) and horseradish-peroxidase-conjugated anti-mouse IgG (GE Healthcare) were used for the detection of GAPDH.
6
Protein Expression Analysis by Western Blot
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A total of 20 μg of IPAs protein were separated on a Hoefer Mini VE system (GE) (Thermofisher), using a precast 8%–25% PhastGel. After, SDS/PAGE proteins were transferred to a nitrocellulose filter (Amersham). Membranes were then probed with rabbit anti-phospho-p38 (Thr180/Tyr182) (Cell Signaling); rabbit anti-p38 (Cell Signaling), rabbit anti-Procaspase 3 (Santa Cruz) and rabbit anti-caspase 3 (Santa Cruz); rabbit anti-nNOS (Cell Signaling); and rabbit anti-α/β-tubulin (Cell Signaling). Immunostaining was detected using horseradish peroxidase-conjugated anti-rabbit IgG (GE). Immunoblots were revealed by the ECL prime (Amersham) and quantitated by densitometry using ImageJ software (NIH).
7
Western Blot Analysis of MYCL and BCL-2
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MYCL and BCL-2, before and after treatment with transduction proteins, were detected by the western blotting methods. The samples were loaded onto a 5–20% gradient polyacrylamide (Wako Co. Ltd, Tokyo, Japan) and electrophoretically transferred to nitrocellulose membranes (GE Healthcare, Danbury, Connecticut, USA). The membranes were blocked with 10% skim milk in PBS. The primary antibodies were anti-MYCL mouse polyclonal antibody (ab167315; Abcam, Cambridge, UK), anti-MYCL rabbit polyclonal antibody (D01P; Abnova, Taipei, Taiwan), anti-BCL2 monoclonal mouse antibody (M0887; Dako, Glostrup, Denmark), anti-FLAG(TM) rabbit monoclonal antibody (F7425; Sigma Aldrich, St Louis, Missouri, USA) and anti-GAPDH(FL-355) rabbit polyclonal antibody (sd-25778; Santa Cruz Biotechnology). The secondary antibodies were horseradish peroxidase-conjugated antirabbit IgG (GE Healthcare). Protein expression was detected using the clarity western ECL substrate (Bio-Rad).
8
Western Blot Analysis of Epigenetic Regulators
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Cell lysates and immunoprecipitants were used for western blot analysis. Proteins were separated by SDS polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Bio-Rad), and probed with primary antibody. The following antibodies were used: anti-SALL3 (Abnova PAB28233, 1:1000), anti-DNMT3A (Abnova 64B1446, 1:1000), anti-DNMT3B (R&D Systems AF7646, 1:1000), anti-LSD1 (Cell Signaling Technology 2184, 1:1000), and anti-β-actin (Sigma-Aldrich A5441, 1:1000). The membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare, 1:5000), anti-mouse IgG (Invitrogen, 1:5000), or anti-sheep IgG (Abcam, 1:5000). Proteins were visualized with ECL Prime Western Blotting Detection Reagent (GE Healthcare) and ChemiDoc Touch Imaging System (BioRad). Uncropped images of scanned blots shown in Supplementary Figure 12 are provided in Supplementary Information file.
9
Macrophage Protein Isolation and Western Blot
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Total protein was isolated from cultured peritoneal macrophages in a solution containing 50 mM Tris-HCL pH 7.4, 1% NP-40, 10% glycerol, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 1 mM DTT, 4.5 mM sodium pyrophosphate, 10 mM β-glycerophosphate, and a protease inhibitor cocktail tablet (Roche Diagnostics). The supernatants were collected after centrifugation at 14,000 rpm at 4 °C for 20 min, and protein content determined with a spectrophotometer using absorption at 570 nm. Forty micrograms of protein was suspended in two volumes of double-strength sodium dodecyl sulfate (SDS) sample buffer (Bio-Rad Laboratories) and subjected to 6–15% SDS–polyacrylamide gel electrophoresis. Proteins were then transferred to polyvinylidene fluoride membranes that were blocked with 10% non-fat dried milk in Tris–HCl-buffered saline containing 0.05% Tween-20. Membranes were immunoblotted overnight at 4 °C with the following primary antibodies: rabbit anti-CD80 (Abcam, ab53003, 1:2000), rabbit anti-CD86 (Abcam, ab53004, 1:2000), and rabbit anti-actin (Sigma, A2006, 1:1000). Bound primary antibodies were visualized using horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare, 1:5000) followed by chemiluminescence detection using an ECL kit (Pierce).
10
Western Blot Analysis of MAPK Phosphorylation
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Microglial cell lysates were boiled after the addition of sample buffer (1 M Tris-HCl, 20% sodium dodecyl sulfate (SDS), and 2.5% glycerol). Fifty micrograms of total protein were separated on a 5 to 20% Tris-glycine SDS-polyacrylamide gel and blotted onto Hybond-P polyvinylidene difluoride (PVDF) membranes (GE Healthcare UK, Buckinghamshire, UK). Membranes were blocked with 1% skim milk in Tris-buffered saline containing 0.05% Tween 20 for 1 h at room temperature. Primary antibodies to detect phosphorylated and total MAPK (Cell Signaling, Danvers, MA, USA) were applied at the concentrations recommended by the manufacturers. The secondary antibody was horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare), which was used at a dilution of 1:1000. SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Rockford, IL, USA) was used according to the manufacturer’s instructions. The intensities of the bands were calculated using the CS Analyzer 1.0 (Atto Corporation, Tokyo, Japan).
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