1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-2000](DSPE-PEG2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[Biotinyl(PolyethyleneGlycol)2000] (DSPE-PEG2000-Biotin) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL,USA). Avidin and DiI (red) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Biotinylated anti-mouse CD309 (FLK1) antibody, anti-mice CD31 (PECAM-1) antibody and fluorescein isothiocyanate (FITC)-labeled anti-rat IgG2a antibody were purchased from eBiosciences (San Diego, CA, USA). Rabbit anti-mouse VEGF receptor 2 antibody and HRP-linked goat anti-rabbit IgG antibody were purchased from Cell Signaling (Cell Signaling Technology Inc., Danvers, MA). Goat anti-rabbit IgG conjugated to Cy3 antibody was obtained from Biorbyt (Biorbyt Limited, Cambridge, UK). All other reagents were of analytical grade. Mice brain microvascular endothelial cells (bEnd.3) were purchased from the American Type Culture Collection (Manassas, VA, USA). Female and male KM mice, weighing about 30 g (8-10 weeks old), were obtained from medical experimental animal center of Guangdong province. (Guangzhou, China).
Goat anti rabbit igg hrp linked antibody
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, Germany
Goat anti-rabbit IgG HRP-linked antibody is a secondary antibody that binds to rabbit primary antibodies. The antibody is conjugated with horseradish peroxidase (HRP), which can be used for detection in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Cell Signaling Technology (5 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (4 mentions)
Protease inhibitor, by Merck Group (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Ai600 imager, by Cytiva (3 mentions)
Anti actin, by Merck Group (3 mentions)
Amersham imager, by Cytiva (3 mentions)
Yl1 2, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
Goat anti mouse igg hrp linked antibody, by Cell Signaling Technology (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Anti top1mt 3, by Developmental Studies Hybridoma Bank (2 mentions)
Clarity ecl substrate, by Bio-Rad (2 mentions)
Oxphos antibody cocktail, by Abcam (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Anti mtco2, by Abcam (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Anti ps375, by Cell Signaling Technology (2 mentions)
Anti mop antibody umb 3, by Abcam (2 mentions)
36 protocols using goat anti rabbit igg hrp linked antibody
1
Formulation and Characterization of Targeted Lipid Nanoparticles
2
Western Blot Analysis of PLK4 and β-Catenin
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The protein level of PLK4 in HCoEpic and CRC cells, as well as nuclear translocation of β-catenin in HCT-116 and LoVo cells after transfection were determined by western blot. Protein of the cells was extracted using a nucleoprotein extraction kit (Sangon Biotech Co., Ltd.) or RIPA reagent (Sangon Biotech Co., Ltd.) and quantified using an enhanced BCA protein assay kit (Beyotime Institute of Biotechnology). Subsequently, the protein (20 µg) was separated using 4–20% SDS-PAGE, followed by transferring onto nitrocellulose membranes (Pall Life Sciences). Then, the membranes were blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) at room temperature for 1 h, and incubated with primary antibodies [β-catenin antibody (Cell Signaling Technology, Inc; 8480; 1:1,000), histone H3 antibody (Cell Signaling Technology, Inc; 4499; 1:2,000), PLK4 antibody (Cell Signaling Technology, Inc; 71033, 1:1,000) and GAPDH (Cell Signaling Technology, Inc; 2118; 1:1,000)] at 4°C overnight, followed by an HRP-linked goat anti-rabbit IgG antibody (Cell Signaling Technology, Inc; 7074; 1:3,000) at room temperature for 1 h. Then, the brands were visualized with ECL-PLUS reagents (Thermo Fisher Scientific, Inc.) and analyzed by ImageJ software (v1.5; NIH).
3
Immunoblotting Antibody Panel for Cellular Analysis
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For the immunoblotting experiment, we used the following antibodies: rabbit monoclonal anti-PARP1 antibody (EPR18461: abcam, Cat#ab191217, 1/5000), mouse monoclonal anti-Actin antibody (AC-40: Sigma, Cat#A3853, 1/10000), rat monoclonal anti-α-tubulin antibody (YL1/2: Santa Cruz Biotechnology, Cat#sc-53029, 1/20000), rabbit polyclonal anti-PAR antibody (Enzo Life Science, ALX-210-890A-0100, 1/5000), rabbit polyclonal anti-NAMPT antibody (BETHYL, Cat#A300-372A, 1/10000), rabbit polyclonal anti-caspase-3 antibody (Cell Signaling, Cat#9662, 1/2000), mouse monoclonal anti-caspase-8 antibody (1C12: Cell Signaling, Cat#9746, 1/2000), mouse monoclonal anti-caspase-9 antibody (C9: Cell Signaling, Cat#9508, 1/2000), rabbit polyclonal anti-AIF antibody (Cell Signaling, Cat#4642, 1/5000), mouse monoclonal anti-Histone H2AX antibody (322105: R&D SYSTEMS, Cat#MAB3406, 1/1000) and rabbit polyclonal anti-γH2AX antibody (abcam, Cat#ab2893, 1/1000). We also used a horse anti-mouse IgG, HRP-linked antibody (Cell Signaling, Cat#7076); a goat anti-rabbit IgG, HRP-linked antibody (Cell Signaling, Cat#7074); and a goat anti-rat IgG, HRP-linked antibody (Cell Signaling, Cat#7077).
4
Western Blot Analysis of SOX9 Protein
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Collected protein lysates by digesting cells in RIPA buffer containing protease inhibitors (Sigma-Aldrich, USA). BCA protein assay kit (Thermo Scientific, USA) was used to evaluate protein concentration. A total of 30 μg protein was electrophoresed on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. Then, the membranes were incubated with specific first antibodies and corresponding second antibody. The specific antibodies were list as below: SOX9 (Cell signaling #82630; 1:1000), GAPDH (Cell signaling #2118; 1:1000). The second antibody was goat anti-rabbit IgG HRP-linked antibody (Cell signaling #7074; 1:4000).
5
Insulin Receptor Signaling in Nrxn1α Mice
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Given the reduce glucose utilization we identified in the mPFC of Nrxn1α+/− mice, and the reduced PFC insulin signaling (Zhao et al., 2006 (link)) and insulin resistance (Manco et al., 2021 (link)) seen in disorders associated with 2p16.3 deletion, we used Western blotting to characterize insulin receptor signaling in the PFC and hippocampus of Nrxn1α+/− mice (WT: n = 6 [male, n = 3]; Nrxn1α+/−: n = 8 [male, n = 4]). The primary antibodies were for the insulin receptor (beta subunit: IRβ, Merck Millipore, Cat. No. 07–724, 1:1000) and the phosphorylated/activated insulin receptor (tyrosine 972 phosphorylated, IR pTyr972/pIR, Merck Millipore, Cat. No. 07–838, 1:1000). The secondary antibody was a goat anti‐rabbit IgG, HRP‐linked Antibody (Cell Signaling, Cat. No. 7074S, 1:2000). Images were acquired with the Chemi‐Doc MP system and analyzed using Image Lab (Bio‐Rad Laboratories Ltd), with normalization to total protein load. Data were then normalized to same‐sex WT control. Full details are provided in the supplemental information.
6
Immunoblotting analysis of STAT signaling
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Cells were collected and lysed in lysis buffer (Thermo Fisher Scientific) after stimulation of mIL-11 (R&D systems), electrophoresed on a 10% SDS-PAGE, and blotted onto PVDF membranes (Biorad). After blocking, the membranes were incubated with primary antibodies overnight at 4 °C, and then with HRP-conjugated secondary antibodies for 1 h. Protein bands were visualized with a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher). The sources for the antibodies were as follows: Rabbit monoclonal antibodies to STAT1, STAT3, p-STAT1, HDAC4, HDAC5, GAPDH (dilution 1:1000). Rabbit polyclonal antibody to p-STAT3 (1:1,000), mouse monoclonal antibody to β-actin (1:1000), goat anti-rabbit IgG HRP-linked antibody, and goat anti-mouse IgG HRP-linked antibody (dilution 1:10,000) are from Cell Signaling (Danvers, MA). Mouse monoclonal antibody to NMP84 (dilution 1:1000) as a nuclear marker was obtained from Abcam, and STAT3-IN-1 from Selleck Biotech Co., Tokyo, Japan.
7
Niclosamide Modulates STAT1/STAT3 Signaling
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Niclosamide (Selleck Chemicals, Houston, TX, USA) was initially diluted in 33% dimethylacetylamine and 67% PEG400 to 25 mmol/L stock concentration. Cells were treated with test compounds at a concentration of 10 μmol/L. Recombinant human IFN-α2a (Novoprotein, Shanghai, China) is produced by Escherichia coli expressing Cys24-Glu188. Interferon was dissolved in distilled water, aliquoted, and stored at a concentration of 2×104 IU/mL. Antibodies used were STAT1 (D1K9Y), Phospho-STAT1 (Tyr701) (58D6), STAT3 (79D7), Phospho-STAT3 (Tyr705) (D3A7) XP®, and β-actin made in rabbits; goat anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA), and GAPDH mouse mAb 39–8600 (ZG003) (ThermoFisher Scientific, Waltham, MA, USA), and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The assay plates (SpectraPlate™-384 TC) were produced by PerkinElmer (Boston, MA, USA).
8
Immunoblotting Analysis of Cellular Fractions
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The nucleus and cytoplasmic fractions of CCA cells were prepared as previously described25 (link). Cell lysates of the entire cells or nucleus fractions were collected by digesting with RIPA buffer and protease inhibitors (Sigma-Aldrich, USA). Protein concentration was determined by BCA kit (Thermo Fisher, USA). A total of 30 μg protein was loaded on 10% or 15% SDS-PAGE gels and transferred onto nitrocellulose membranes. The membranes were incubated with specific first antibodies and corresponding second antibody. The specific antibodies were listed below: Cleaved Caspase-3 #9664, PCNA #13110; MMP-3 #14351, ZEB1 #70512, E-Cadherin #3195, Vimentin #5741, Oct-4 #2750, LIN28A #3695, Nanog #3580, Sox2 #3579, β-Catenin #8480, Phospho-β-Catenin (Ser33/37/Thr41) # 9561, Phospho-GSK-3β (Ser9) #5558, GSK-3β#12456, c-Myc #9402, Survivin #2803, Lamin B1 #12586, and GAPDH #5174 were purchased from Cell Signaling Technology with a work concentration at 1: 1000. BCL9L #ab233736 was purchased from Abcam with a work concentration at 1: 500. The second antibodies were goat anti-rabbit IgG HRP-linked antibody (Cell signaling #7074; 1:4000) and sheep anti-mouse IgG-HRP (GE/Amershan #NXA931; 1: 5000).
9
Western Blot Analysis of EMT Markers
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Cells were lysed in RIPA buffer containing protease inhibitors (Sigma-Aldrich, Carlsbad, CA, USA). BCA protein assay kit (Thermo Scientific, Grand Island, NY, USA) was used to quantify protein concentration. A total of 30 ug protein was electrophoresed by 10% SDS-PAGE and transferred onto nitrocellulose membranes, then incubated with specific first antibodies overnight and corresponding second antibodies for 1 hr. The specific first antibodies were list as follows: SRC-1 (128E7) Rabbit mAb (Cell signaling #2191; 1: 1000); Anti-Twist antibody (Abcam #ab50581; 1: 2000), Vimentin (D21H3) XP Rabbit mAb (Cell signaling #5741; 1: 1000); E-Cadherin (24E10) Rabbit mAb (Cell signaling #3195; 1: 1000). The second antibody was goat anti-rabbit IgG HRP-linked antibody (Cell Signaling #7074; 1: 4000).
10
Western Blot Analysis of Gankyrin and GAPDH
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Western blot was performed as we previously reported [19 (link)]. The following primary antibodies were used: rabbit anti-gankyrin antibody (Abcam, USA), rabbit anti-GAPDH antibody (Cell Signaling Technology, USA). The secondary antibodies were goat anti-rabbit IgG-HRP-linked antibody from Cell Signaling Technology (Danvers, MA, USA).
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