RIPA buffer (EBA-1149, Elpis Biotech Inc., Seoul, Republic of Korea) containing 5 μg/mL of aprotinin and leupeptin were used to extract proteins from colon tissues and Caco-2 cells. Protein lysates were obtained as supernatant collection after centrifugation for 20 min at 4 °C, 13,000 rpm. Lysates (40 μg) was separated by 8–14% gel electrophoresis and were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were subjected to blocking in 5% skim milk in TBST (Tris-buffered saline with 0.1% Tween 20) before being incubated overnight with primary antibodies overnight against FN (1:500), α-SMA (1:500), E-cadherin (1:1000), TGF-β R1 (1:500), C/EBPβ (1:500), Smad2/3 (1:500), and GAPDH (1:1000) at 4 °C. The membranes were then incubated for 1 h with a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Millipore, Billerica, MA, USA) before the protein bands were identified using an ImageQuantTM LAS 400 mini (GE Healthcare, Buckinghamshire, UK). The protein bands were visualized by using ECL™ Select Western blotting detection reagent (Cytiva, Buckinghamshire, UK). ImageJ analysis (National Institutes of Health, Bethesda, MD, USA) was used to quantify the bands. As an internal control, GAPDH was employed.
Horseradish peroxidase conjugated goat anti mouse secondary antibody
Manufactured by Merck Group
Sourced in United States, Germany
The Horseradish peroxidase-conjugated goat anti-mouse secondary antibody is a laboratory reagent used in various immunoassay techniques. It contains a horseradish peroxidase enzyme conjugated to a goat-derived antibody that specifically binds to mouse primary antibodies. This product can be used to detect and visualize the presence of target molecules in samples.
Automatically generated - may contain errors
Lab products found in correlation
Ecl select western blotting detection reagent, by Cytiva (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Mouse anti α tubulin primary antibody, by Merck Group (2 mentions)
Water soluble unesterified cholesterol, by Merck Group (2 mentions)
Digitonin, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Carl Roth (2 mentions)
Apolipoprotein apo a1, by Merck Group (2 mentions)
Rpmi 1640 medium without l glutamine, by Lonza (2 mentions)
Hrp linked anti rabbit igg secondary antibody, by New England Biolabs (2 mentions)
3h cholesterol, by PerkinElmer (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (2 mentions)
Resazurin sodium salt, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Eba 1149, by Elpis Biotech (1 mentions)
Polyvinylidene difuoride membrane, by Merck Group (1 mentions)
Image quant las400 mini, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Bio-Rad (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
16 protocols using horseradish peroxidase conjugated goat anti mouse secondary antibody
1
Western Blot Analysis of Colon Tissue and Caco-2 Cells
2
Western Blot Analysis of Renal Cell Proteins
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HK-2 cells and renal tissues were lysed for 1 h with RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing 5 μg/mL of aprotinin and leupeptin. After centrifugation at 4 °C and 15,814 x g for 20 min, the supernatant was collected and used as protein lysates. The protein concentrations were examined by BCA protein assay kit (Thermo Fisher Scientific). Subsequently, 40 µg of lysates were separated using 8–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to polyvinylidene difuoride membranes (Millipore, Billerica, MA, USA). After blocking using 4–5% skim milk in Tris buffered saline-0.05% (v/v) Tween-20 for 30 min, the membranes were incubated with primary antibodies overnight against HIF-1α (1:500), FN (1:800), α-SMA (1:300), E-cadherin (1:1000), ATF-4 (1:500), GRP78 (1:500), and GAPDH (1:1000) at 4 °C. After that, goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Millipore, Billerica, MA, USA) was used to incubate the membranes at 25 °C for 1 h, and protein bands were detected with ECL™ Select western blotting detection reagent (Cytiva, Buckinghamshire, UK). The images were analyzed with ImageQuant™ LAS 400 mini (GE Healthcare, Buckinghamshire, UK), which were then quantified by ImageJ analysis (National Institutes of Health, Bethesda, MD, USA). GAPDH was used as an internal control.
3
Western Blot Analysis of iNOS in Microglia
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Primary microglia were lysed in a buffer containing 50 mM Tris–HCl, pH 7.2, 0.1% sodium deoxycholate, 1% Triton X-100, 5 mM EDTA, 5 mM EGTA, 150 mM NaCl, 40 mM NaF, 2.175 mM NaVO4, 0.1% SDS, 0.1% aprotinin and 1 mM PMSF. Ten micrograms of protein underwent SDS–PAGE following transfer on nitrocellulose membranes. Bands were detected using Pierce™ ECL Western Blotting Substrate (ThermoFisher Scientific, Waltham, MA, USA) on a Chemidoc digital imaging machine. We utilized an anti-iNOS rabbit primary antibody (1:200 in 1% BSA; Cayman Chemical, Ann Arbor, MI, USA) followed by a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:3,000; Bio-Rad, Hercules, CA, USA). Internal loading control was performed by anti-α-Tubulin mouse primary antibody (1:500 in 1% BSA; Sigma-Adrich, St. Louis, MO, USA) followed by a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:1,000; Sigma-Aldrich, St. Louis, MO, USA; see Supplementary Figure S2 for antibody specificity). Densitometry quantification was performed with ImageJ Software (NIH) and expressed as ratio of iNOS to α-tubulin.
4
Antibody Characterization Protocol
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Rabbit anti-TET1 (Millipore, 09–872), mouse anti-TET1 (Active Motif, 91171), rat anti-TET1 (Active Motif, 61741, refer to as 5D6), mouse anti-HA (Invitrogen, 26183), mouse anti-Flag (sigma, F1804 and F7425), mouse anti-Tuj1 (Biolegend, 801201), rabbit anti-GFAP (Sigma, HPA056030) antibodies were purchased commercially. Rabbit anti-EGR1 antibody (sc-189), mouse anti-EGR1 antibody (sc-101033), rabbit normal IgG (sc-2027) and mouse normal IgG (sc-2025) were purchased from Santa Cruz Biotechnology. For western blot analysis, goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Invitrogen, 65–6120) was used at a 1:5000 dilution, goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Sigma, A8924) was used at a 1:10,000 dilution. The antibodies used for the IP and western blot experiments were summarized in Supplementary Data 6 .
5
Curcumin-loaded Nanoparticles Induce Apoptosis in HeLa Cells
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HeLa cells (1 × 106 cells per well in 6-well plate) treated with curcumin-loaded NPs (0.005 and 0.01 mg/mL of curcumin-loaded NPs) for 72 h were washed with PBS and then lysed in a buffer containing 50 mM Tris–HCl, pH 7.2, 0.1% sodium deoxycholate, 1% Triton X-100, 5 mM EDTA, 5 mM EGTA, 150 mM NaCl, 40 mM NaF, 2.175 mM NaVO4, 0.1% SDS, 0.1% aprotinin and 1 mM PMSF. Forty micrograms of protein underwent SDS–PAGE following transfer on PVDF membranes (Amersham™ Hybond® P, Dasser Germany). Bands were detected using Immobilon ECL Western Blotting Substrate (ThermoFisher Scientific, Waltham, MA, USA) on a Chemidoc digital imaging machine (Bio-Rad). An anti-Cleaved Caspase-3 primary antibody (1:1000 in 1% BSA, Cell Signaling Technology, Inc. MA, USA) and an anti-PARP-1 primary antibody (1:1000 in 1% BSA, Santa Cruz Biotechnology, Dallas, TX, USA), were used both followed by a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:10,000; Abcam Cambridge CB2 0AX UK). Internal loading control was performed using the anti-α -Tubulin mouse primary antibody (1:10,000 in 1% BSA; Sigma-Aldrich) followed by a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:10,000, Sigma-Aldrich). Densitometry quantification was performed with ImageJ Software (NIH) and expressed as ratio of cleaved caspase-3 and PARP to α-tubulin.
6
Protein Extraction and Western Blot Analysis of Agroinfiltrated N. benthamiana
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Leaves of N. benthamiana infiltrated with A. tumefaciens were collected at 2 dpi and the total protein was extracted using extraction buffer (100 mM Tris‐HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 mM dithiothreitol, 0.5% Triton X‐100, 2% polyvinylpolypyrrolidone, and 1× Roche Complete protein inhibitor tablets). A western blot assay was performed with the samples as described previously (Wang et al., 2018 ). Membranes were probed with primary mouse anti‐His or anti‐GFP tag monoclonal antibody (1∶3000) (Sigma‐Aldrich), followed by goat anti‐mouse horseradish peroxidase‐conjugated secondary antibody (1∶10,000). The protein bands were detected using a BeyoECL STAR Western kit (Beyotime) following the manufacturer's instructions.
7
Cholesterol Trafficking Assay Protocol
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Pioglitazone was bought from Molekula (Munich, Germany) and [3H]-cholesterol (1 mCi, 37 MBq) from Perkin Elmer Life Sciences (Vienna, Austria). Digitonin was purchased from Sigma-Aldrich via Fluka (Vienna, Austria). Bovine serum albumin (BSA) was obtained from Roth (Karlsruhe, Germany). Resazurin sodium salt, water soluble unesterified cholesterol, apolipoprotein (apo) A1, and phorbol 12-myristate 13-acetate (PMA) were provided by Sigma-Aldrich.
Phenol red Roswell Park Memorial Institute (RPMI) 1640 medium withoutl -glutamine was purchased from Lonza (Basel, Switzerland). Fetal bovine serum (FBS) was from Gibco (Lofer, Austria).
Primary antibody against ABCA1 was provided by Novus Biologicals (Vienna, Austria). The anti-actin antibody was obtained from MP biologicals (Illkirch, France). HRP-linked anti-rabbit IgG secondary antibody was acquired from New England Biolabs (Frankfurt, Germany) and horseradish peroxidase conjugated goat anti-mouse secondary antibody from Upstate (Millipore, Vienna, Austria). All antibodies were diluted 1:500 except the anti-mouse secondary antibody (1:10,000).
Phenol red Roswell Park Memorial Institute (RPMI) 1640 medium without
Primary antibody against ABCA1 was provided by Novus Biologicals (Vienna, Austria). The anti-actin antibody was obtained from MP biologicals (Illkirch, France). HRP-linked anti-rabbit IgG secondary antibody was acquired from New England Biolabs (Frankfurt, Germany) and horseradish peroxidase conjugated goat anti-mouse secondary antibody from Upstate (Millipore, Vienna, Austria). All antibodies were diluted 1:500 except the anti-mouse secondary antibody (1:10,000).
8
Membrane Protein Isolation and Analysis
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HCE cells were washed with cold phosphate-buffered saline (PBS), collected, and lysed with buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM dithiothreitol, 0.32 M sucrose, and a protease inhibitor cocktail) to obtain membrane fractions. The HCE cell lysates were centrifuged at 12,000 × g for 30 min at 4℃; the pellets were resuspended in the same lysis buffer (1% Triton X-100, incubated on ice for 45 min) and centrifuged again at 12,000 × g for 30 min at 4℃. The supernatants were collected and used as membrane fractions. Total cell protein was measured by the BCA method (Thermo Scientific, USA). HCE cell membrane fractions were separated by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and then transferred to polyvinylidene difluoride membranes. After that, horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Millipore, USA) and 16B12 monoclonal antibody (Millipore) were added, and the results were visualized by enhanced chemiluminescence assay (GE Healthcare, UK).
9
Cholesterol Trafficking Assay Protocol
10
Histological Analysis of Omentum and Pancreas
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At the end of 28 days, rats were anesthetized and transcardially perfused with saline and formalin, respectively. Omentum and pancreas tissues were collected and fixed in formalin for approximately 18 h before being embedded into paraffin. Tissues were sectioned with a Leica microtome at 5 lm thickness. Sections were deparaffinized in xylene and rehydrated in serial ethanol series for hematoxylin & eosin (H&E) staining and Masson's trichrome staining according to the standard protocol. For immunohistochemistry experiments, sections were stained with anti-insulin (1:500; Sigma I2018, clone K36AC10), anti-macrophages/monocytes (1:300; Millipore, MAB1435, clone ED-1) and anti-von Willebrand Factor (1:200; Abcam, ab6994) antibodies. After primary antibody staining, horseradish peroxidase conjugated goat anti-mouse secondary antibody (1:500; Millipore) was used followed by 3,3 0 -diaminobenzidine (DAB) staining. All samples were mounted onto glass slides using xylene based mounting medium. Digital images were acquired via Zeiss Axio Scope A1. For vascular density quantification, each paraffin-embedded omentum tissue was serially sectioned and 5 matching slides were selected from each tissue for quantification with approximately 100 lm intervals. Images were acquired by using a 20Â objective.
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