Samples were separated by TGX stain-free precast gels (4-15% gradient, Bio-Rad), ultraviolet-activated with ChemiDoc Touch system (Bio-Rad) for direct imaging of total proteins and then transferred to nitrocellulose membranes for immunoblotting analysis. After blocking with 5% non-fat dried milk in Tris-buffered saline Tween-20 (TBST; 28 mM Tris, 136.7 mM NaCl, 0.05% Tween-20, pH 7.4) for 45 min at room temperature, membranes were incubated with rabbit anti-Nlg1 (129013, Synaptic systems) diluted at 1:1,000 with 0.5% non-fat dried milk in TBST, followed by horse radish peroxidase-conjugated anti-rabbit antibody (Jackson ImmunoResearch) for 1 h at room temperature. Target proteins were detected by chemiluminescence with Super signal West Dura (Pierce) on the ChemiDoc Touch system (Bio-Rad). The theoretical molecular weight of Nlg1 is 93 kDa, but the apparent molecular weight in immunoblots is ∼130 kDa, likely due to glycosylations65 (link). For quantification, the intensity of the chemiluminescence signal of each lane was normalized by the total protein signal on the same lane, revealed by the stain-free technology.
Horseradish peroxidase conjugated anti rabbit antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Horseradish peroxidase-conjugated anti-rabbit antibody is a secondary antibody that binds to rabbit primary antibodies. The horseradish peroxidase enzyme conjugated to the antibody can be used to detect and visualize the presence of the rabbit primary antibody in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Anti mouse antibody, by Jackson ImmunoResearch (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Chemidoc touch system, by Bio-Rad (1 mentions)
Tgx stain free precast gel, by Bio-Rad (1 mentions)
Ab96599, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti mouse antibody, by Agilent Technologies (1 mentions)
Sc 154, by Santa Cruz Biotechnology (1 mentions)
Anti h3 k4 trimethyl, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Gel pro analyzer software 4, by Media Cybernetics (1 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Timp 1, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Skim milk solution, by BD (1 mentions)
Image lab 3, by Bio-Rad (1 mentions)
Bovine serum albumin solution, by Merck Group (1 mentions)
Ecl kit, by GenDEPOT (1 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
12 protocols using horseradish peroxidase conjugated anti rabbit antibody
1
Immunoblotting of Neuroligin-1 Protein
2
Synthesis and Characterization of Masitinib and Derivatives
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Masitinib (99% pure) and masitinib-NH2 (99% pure) were provided by AB Science S.A. Masitinib-NH2-LC-Biotin was synthesised from masitinib-NH2 (for synthesis and characterisation, see Supplementary Note 1 , Supplementary Fig. 14 ). DI-39 was synthesised as described previously39 (link), 40 (link). Protein kinase inhibitors were purchased from Selleckchem. Reagents and substrates for kinetic assays were all purchased from Sigma-Aldrich. Each reagent was obtained as powder and dissolved in H2O or DMSO and stored as aliquots at −20 °C. Fresh dilutions were prepared for each experiment. Primary antibodies used were a rabbit polyclonal anti-deoxycytidine kinase antibody (ab96599, Abcam, 1:1000), a rabbit polyclonal anti-ERK2 antibody (sc-154, Santa Cruz Biotechnology, 1:2000), a rabbit polyclonal anti-AKT1 antibody (#9272, Cell Signalling Technology, 1:1000), a mouse monoclonal anti-Lyn antibody (610004, BD Biosciences, 1:1000), and a rabbit polyclonal anti-Kit antibody (#3074, Cell Signalling Technology, 1:1000). Primary antibodies were detected using 1:20,000 horseradish peroxidase-conjugated anti-rabbit antibody (Jackson Immunoresearch Laboratories Inc.) or 1:20,000 horseradish peroxidase-conjugated anti-mouse antibody (Dako).
3
Histopathological Analysis of Lung Tissues
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Upper lobes of left lungs were fixed in 10% buffered formalin and sections of 5 μm in thickness from formalin fixed and paraffin embedded tissues were cut onto glass slides. H&E staining for pathohistological analysis was performed by the Department of Histopathology (Air Force Medical University, China). H3K4me3 expression was determined by immunohistochemistry (IHC), and IHC was performed by Shenyang Wanleibio Biotechnology Limited Company (China). Antibodies used in IHC were polyclonal rabbit anti-H3-K4 trimethyl (1:500, Abcam) and horseradish peroxidase-conjugated anti-rabbit antibody (1:500, Jackson Immunoresearch Laboratories).
4
Protein Expression Profiling of MMPs and TIMPs
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Cytosol and nuclear section of proteins were prepared with the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) in accordance with the manufacturer's protocols, and were quantified by the BCA Protein Assay Reagent Kit (Pierce). Equal amounts (30 μg) of samples were separated by SDS/12% PAGE and then were electrotransferred to nitrocellulose membrane (Millipore). The membranes were blocked with 2% BSA (Sigma–Aldrich) in PBS (4°C overnight), and then were incubated for 2 h at room temperature with specific primary antibody, against MMP-1, -3, -9, TIMP-1 or GAPDH (all from Abcam), against NF-κB/p65 or IκBα (both from Cell Signaling Technology). Then membranes were incubated with horseradish peroxidase-conjugated anti-rabbit antibody (Jackson Immuno Research) for 1 h at room temperature and the blots were detected using Enhanced Chemiluminescence reagent (Amersham Pharmacia Biotech).The integral optical density (IOD) of target band was analysed with Gel-Pro Analyzer Software 4.0 (Media Cybernetics), and was presented as a relative level of target protein IOD / GAPDH IOD.
5
Western Blot Analysis of Cardiac Protein
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The halves of the hearts were homogenized, and total protein was extracted using protein lysis buffer (Pro-prep; iNtRON, Seongnam, Korea). Cardiac tissue samples containing 60 μg total protein and H9c2 cell extracts containing 10 μg total proteins were boiled for 10 minutes and loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (8% stacking and 10%, 15% separating gels). Separated proteins were transferred to nitrocellulose membranes (NC, 0.45 μm pore size; Bio-Rad, Hercules, CA, USA) or Iimmobilon-P transfer membrane (PVDF, 0.45 μm pore size; Millipore, Billerica, MD, USA). After blocking in 5% bovine serum albumin solution (Sigma-Aldrich) or 5% skim milk solution (BD Biosciences, San Diego, CA, USA) for 60 minutes, the membranes were incubated with primary antibody overnight at 4°C. The primary antibodies used are specified in Supplementary Table 1 . Blots were incubated with horseradish peroxidase-conjugated anti-rabbit antibody (1:2,000; Jackson Immunoresearch, West Grove, IA, USA) or anti-mouse antibody (1:2,000; Jackson Immunoresearch) for 1 hour at room temperature. Glyceraldehyde-3-phosphate dehydrogenase was used as a protein loading control. Positive protein bands were visualized using an ECL kit (GenDEPOT, Barker, NY, USA), and results were quantified with an image analyzer (Image lab 3.0; Bio-Rad).
6
Western Blot Analysis of Inflammatory Proteins
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Five to ten micrograms of protein from the cell lysates was run on 4 to 12% NuPAGE Bis-Tris SDS-PAGE gels (Invitrogen by ThermoFisher Scientific) and transferred to polyvinylidene difluoride membranes (ThermoFisher Scientific) using an iBlot 2 gel transfer device (Life Technologies). Membranes were blocked in 5% nonfat dairy milk and incubated with primary antibodies overnight. The primary antibodies used are rabbit-anti-mouse monoclonal total pyrin antibody (ab195975; abcam), rabbit-anti-mouse monoclonal antibody phospho-serine 205 (ab201784; abcam), rabbit-anti-mouse/human IL-1β (number 12242; Cell Signaling), and rabbit-anti-mouse/human polyclonal β-actin (number 4967; Cell Signaling). Horseradish peroxidase-conjugated anti-rabbit antibody (Jackson Laboratory) was used as a secondary antibody. Proteins were visualized using chemiluminescent detection reagent (GE Healthcare) on an iBright FL1500 (ThermoFisher Scientific).
7
Notch Pathway Protein Detection
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Immunohistochemistry and western blots were done as routine, using primary antibodies including rabbit anti-Jagged1, anti-Notch3, anti-NICD (Notch intracellular domain), anti-HES1 and anti-β-actin (ab7771, ab23426, ab8925, ab108937 and ab8227; Abcam, Cambridge, MA, USA). The secondary antibody used a horseradish-peroxidase-conjugated anti-rabbit antibody (Jackson ImmunoResearch Labs, West Grove, PA, USA). The images were taken using microscopes (Nikon, Tokyo, Japan).
8
Quantitative Western Blot Analysis of Adipose Tissue
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Proteins were extracted from mice adipose tissue in 1 × SDS lysis buffer (150 mM NaCl, 25 mM Tris-HCl, pH 7.6, 1% sodium deoxycholate, and 1% NP-40) with a protease inhibitor cocktail (Roche, Switzerland). Protein expression in mice retroperitoneal and epididymal adipose tissues were measured by Western blot with antibodies obtained from Abcam (MCP-1; 1 : 300), Santa Cruz Biotechnology (PPARγ; 1 : 2000), Earth Ox (β-actin; 1 : 1000), and Cell Signaling Technology (TNF-α; 1 : 500 & GAPDH; 1 : 600). Protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked with 5% fat-free milk and incubated with different primary antibodies at 4°C. The bound antibodies were detected using horseradish peroxidase-conjugated anti-rabbit antibodies or anti-mouse antibodies (Jackson Laboratory, ME, USA; 1 : 5,000) for 1.5 h at room temperature. Protein levels were detected with SuperSignal West Pico ECL buffer (Thermo Fisher, IL, USA) using ChemiDoc XRS imaging system (BioRad, CA, USA). The intensity of protein bands was quantitated using Image J analysis software (Version 1.50i; MD, USA).
9
Western Blot Analysis of CSF Proteins
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For western blot analysis, CSF was included in the sample buffer (100 mM Tris–HCl pH 6.8, 4% SDS, 2% bromophenol blue, 20% glycerol, 0.5% β-mercaptoethanol) and heated at 95 °C for 5 min to denature the proteins. Electrophoresis was performed in 10% polyacrylamide gels at 35 mA. Proteins were transferred to a PVDF membrane (Millipore) at 400 mA for 90 min in transfer buffer (39 mM glycine, 48 mM Tris-base pH 8.3, 0.037% SDS, 10% methanol) in a Mini-Trans Blot (Bio-Rad) system. After 1 h of blocking with 5% dehydrated milk in tris-saline buffer (TBS), membranes were incubated overnight with primary antibodies (Table 3 ) at 4 °C. Protein concentration was detected with chemiluminescence (Amersham Biosciences) after one-hour incubation with horseradish peroxidase-conjugated anti-rabbit antibodies (Jackson Immuno Research Laboratories) (Table 3 ). chemiluminescence was detected with ChemiDoc MP, (Biorad) and analyzed with ImageLab software.
10
Western Blot Analysis of IL-2Rα in VSMC and T Cells
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Whole cell lysates from primary VSMCs and Jurkat T cells (ATCC, Manassas, VA) were prepared by lysing and sonicating pelleted cells in radio immuno-precipitation (RIPA) buffer (150 mM NaCl, 25 mM Tris-HCl pH 7.6, 1% Nonidet P40, 1.0% sodium deoxycholate, 0.1% SDS). Splenocytes were separated into membrane, cytoplasmic, and nuclear fractions per manufacturer’s instructions (Cell Fractionation Kit, Cell Signaling Technology, Danvers, MA). Extracts were then separated by SDS-PAGE, using 30 μg protein measured by bicinchoninic acid assay (Pierce, Thermo-Fisher), and transferred to a polyvinylidene difluoride membrane. Some blots, as indicated in figure legends, were assessed for total protein using either stain-free gels (Bio-Rad, Hercules, CA) or the Revert™ 700 Total Protein Stain (LI-COR) prior to blocking. After incubation with 5% nonfat milk in TBST for 60 min, the membrane was incubated with antibodies against IL-2Rα at 4°C for 18 h. Membranes were washed three times for 10 min and incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated anti-rabbit antibodies (Jackson ImmunoResearch, West Grove, PA) at room temperature for 2 hrs. Blots were washed with TBST three times and developed with the ECL system (Luminata Crescendo, Millipore Sigma) according to the manufacturer’s instructions.
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