To measure protein levels, immunoblot analysis was conducted based on a previously published protocol [6 (link)]. The samples were lysed using RIPA buffer (#P0013B, Beyotime Biotechnology, Shanghai, China). An equal amount of protein (20 mg) was loaded onto an SDS-polyacrylamide gel and transferred to the polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 4% bovine serum albumin (BSA, #ST023, Beyotime Biotechnology), followed by overnight incubation with primary antibodies at 4 °C. Afterwards, the membranes were incubated with appropriate secondary antibodies. After washing, the membranes were visualized using ECL (#P0018FS, Beyotime Biotechnology). The antibodies contained anti-NRF2 (1:1000, #16396-1-AP, Proteintech, Wuhan, China), anti-GAPDH (1:10000, #10494-1-AP, Proteintech, Chicago, IL, USA), anti-KEAP1 (1:1000, #80744-1-RR, Proteintech), and HRP-conjugated anti-rabbit IgG antibody (#7074, Cell Signaling Technology, Danvers, MA, USA).
Hrp conjugated anti rabbit igg antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The HRP-conjugated anti-rabbit IgG antibody is a secondary antibody that binds to rabbit primary antibodies. It is conjugated with horseradish peroxidase (HRP), an enzyme that can be used as a reporter for colorimetric or chemiluminescent detection in various immunoassays and immunohistochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated anti mouse igg antibody, by Cell Signaling Technology (12 mentions)
Gapdh, by Cell Signaling Technology (6 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (3 mentions)
Dc protein assay kit, by Bio-Rad (3 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Hrp conjugated monoclonal mouse antiactin antibody, by Merck Group (3 mentions)
Anti mouse igg antibody, by Cell Signaling Technology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Pierce ecl plus, by Thermo Fisher Scientific (2 mentions)
Anti h3k27ac antibody, by Cell Signaling Technology (2 mentions)
Anti total h3 primary antibody, by Cell Signaling Technology (2 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
P akt ser473, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
58 protocols using hrp conjugated anti rabbit igg antibody
1
Quantifying Protein Levels via Immunoblotting
2
Western Blot Analysis of Proteins
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Total cell lysates were prepared in RIPA lysis buffer (Beyotime Biotechnology) supplemented with 1 mM PMSF and 1× protease inhibitor cocktail (Yeasen), and nuclear protein was extracted by nuclear protein extraction kit (Solarbio) in accordance with the manufacturer’s instructions. The samples were separated by SDS-PAGE and transferred to the PVDF membrane (Millipore). The membranes were incubated with the indicated primary antibodies at 4 °C overnight and then incubated with an HRP-conjugated anti-rabbit IgG antibody (Cell Signaling, Cat# 7074, 1:5000) or anti-mouse IgG antibody (Cell Signaling, Cat# 7076, 1:5000) at room temperature for 1 h. The signal was visualized by using the NcmECL Ultra kit (New Cell & Molecular Biotech) and then was exposed by using MiniChemi (SageCreation).
3
Adiponectin-Mediated Cell Signaling Analysis
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Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Thermo Scientific, Waltham, MA, USA. Hank's balanced salt solution (HBSS), rat recombinant globular adiponectin (gAd), Z-VAD-FMK, necrostatin-1, dihydroethidium, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide kit were purchased from Sigma-Aldrich, St. Louis, MO, USA. Antibodies against cleaved caspase-3, caspase-3, RIP1, RIP3, NF-κB, p38MAPK, phosphorylated-NF-κB, phosphorylated-p38MAPK, Bcl-2, Bax, and GAPDH as well as HRP-conjugated anti-rabbit IgG antibody were obtained from Cell Signaling Technology, Inc., Danvers, MA, USA.
4
Histone H3K27 Acetylation Profiling
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Protein was extracted from cells using RIPA buffer (89900, Thermo Fisher Scientific). A total of 60 µg of protein (from whole cell extract) was separated by electrophoresis in a 4–15% precast protein gel (4561086, BioRad) and transferred to PVDF membranes. Blocking was subsequently performed with 5% non-fat milk in TBST, followed by incubation with anti-H3K27Ac antibody at 1:500 dilution (8173S, Cell Signaling Technology) overnight. After 5 washes with TBST, membranes were incubated with HRP-conjugated anti-Rabbit IgG antibody at 1:1000 (7074 Cell Signaling Technology) for 1 h. Pierce ECL Plus (32132, Thermo Fisher Scientific) was used to detect protein bands. Blots were then stripped (46430, Thermo Fisher Scientific) and re-probed with anti-total H3 primary antibody at 1:1000 dilution (14269S, Cell Signaling Technology) as a loading control. HRP-conjugated anti-Mouse IgG antibody (7076, Cell Signaling Technology) was used to detect total H3 signal. Densitometry analysis was performed with image J.
5
Western Blot Analysis of MOR Protein
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The skin samples were lysed in RIPA buffer and then centrifuged at 12,000 rpm for 10 min at 4°C. The proteins in the lysates were detected using a bicinchoninic acid (BCA) protein assay kit following the manufacturer's guidelines. For each sample, 100 μg of protein was separated on an SDS-PAGE gel and then transferred onto nitrocellulose (NC) membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 5% BSA for 2 h at room temperature, then incubated with the primary antibodies, MOR, after which they were washed in three changes of PBS. The membranes were then incubated with secondary HRP-conjugated anti-rabbit IgG antibody (1 : 200 dilution, Cell Signaling Technology, #4412) for 1 h at room temperature. Proteins were visualized by adding ECL (Thermo), then scanning and imaging the membranes using the FluorChem FC3 system (ProteinSimple, USA).
6
Plasma POSTN Quantification by Dot Blot
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Plasma samples from the same patients were tested for POSTN by dot blot assay. For this purpose, 2 ml of blood collected in vacuum tubes containing EDTA was centrifuged at 3000g for 10 minutes. Plasma, obtained by supernatant centrifugation at 15,000g for 10 minutes to eliminate cells and debris, was aliquoted and stored at −80°C. For dot blot assay, 2 μl of plasma was spotted onto a nitrocellulose membrane; dried in air for 1 hour at ambient temperature; and blocked with 0.15 M NaCl, 10 mM Tris–HCl (pH 7.4), and 0.1% Tween-20 solution containing 3% nonfatty milk. After overnight incubation at 4°C with anti-POSTN (1:1000, Acris, Germany), membrane was washed and incubated with HRP-conjugated anti rabbit IgG antibody (1:2000, Cell Signaling Technology Inc., USA). The signal was revealed using the Immobilon Western Chemiluminescent kit (Millipore Corp., USA). After x-ray scanning with GS800 scanner (BioRad, USA), background subtraction, normalization with positive control spots, and densitometric evaluation of each spot were carried out with Quantity One software (BioRad, USA). Comparison of signal intensity, expressed as OD values, was used to determine relative differences in plasmatic levels of POSTN. More detailed information about the POSTN dot blot assay is provided in the supplementary file.
7
Protein Metabolism Regulation in Large Yellow Croaker
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The phosphorylation levels of mTOR and 4-EBP were measured to determine influences of FM replaced by FSM on protein metabolism in the muscle of large yellow croaker. The detailed procedures were according to the previous study by Chen et al. [23 (link)]. The primary antibodies are anti-rabbit mTOR (1 : 1000, #2972, Cell Signaling Technology; MA, USA), anti-rabbit phospho-mTOR (Ser2448) (1 : 1000, #5536, Cell Signaling Technology; MA, USA), anti-rabbit 4E-BP1 (1 : 1000, #9452, Cell Signaling Technology; MA, USA), anti-rabbit phospho-4E-BP1 (Thr37/46) (1 : 1000, #2855, Cell Signaling Technology; MA, USA), anti-mouse β-actin (1 : 5000, AC004, ABclonal Technology; Wuhan, China), as well as anti-rabbit β-tubulin (1 : 5000, AC008, ABclonal Technology; Wuhan, China), respectively. The secondary antibodies are HRP-conjugated anti-rabbit IgG antibody (1 : 10000, 7074, Cell Signaling Technology; MA, USA) and HRP-conjugated mouse anti-rabbit IgG antibody (1 : 10000, 5127, Cell Signaling Technology; MA, USA). Quantification of protein bands was accomplished by the ImageJ software.
8
Protein Expression Analysis of CPE-Treated Cells
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The whole cell lysate from cells treated with CPE or vehicle control was isolated using MPER reagent along with inhibitors (1% phosphatase inhibitor, 1% protease inhibitor and 1% PMSF). BCA Assay was performed for measurement of protein concentration in the cell lysates. Equal amount of protein sample from vehicle control and CPE‐treated cells was separated using 12% or 15% SDS‐PAGE and then electrotransferred on to a PVDF membrane. The membranes were incubated (at 4°C) overnight, with primary antibody specific for NF‐κB, oxidative stress markers, LC3B, Beclin‐1, p62, BAD, Bcl‐2 (Abcam), BAX, p27 (Cell Signaling Technology) or GAPDH (Santa Cruz Biotechnology), followed by corresponding HRP conjugated anti‐rabbit IgG antibody (Cell Signaling Technology) or anti‐mouse IgG antibody (Cell Signaling Technology) incubation for 2 h. After that, the membranes were thoroughly washed in TBST (1×). Finally, the PVDF membrane was probed with ECL solution (Thermo Fisher) and observed in ChemiDoc Imaging System (Azure Biosystems) to visualize the protein bands.
9
Western Blot Analysis of Cytoskeletal Proteins
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Cells were lysed in RIPA Buffer (Sigma-Aldrich) and protein concentration was determined by using the BCA assay kit (Sigma-Aldrich). Total protein (40 μg) was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% or 5% gel and electrotransferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were then incubated in 5% nonfat dry milk in Tris-buffered saline (TBS, pH 7.6) and then overnight at 4 °C with a rabbit monoclonal anti-DHC2 (#ab122525; Abcam) at a dilution of 1:1000, anti-KIF2B (#ab98214; Abcam) at a dilution of 1:2000 or anti-Phospho-H2A.X (#9718; Cell Signaling Technology) at a dilution of 1:1000. Membranes were then incubated at 37 °C for 1 h with an HRP-conjugated anti-rabbit IgG antibody (#7074; Cell Signaling Technology) at a dilution of 1:2000. After three washes with TBS, membranes were briefly incubated with chemiluminescent HRP substrate (#WBKLS0100; Millipore) and were photo-developed in Image Station 2000 MM (Kodak, Rochester, Minnesota, USA). Quantity One 4.6.2 (Bio-Rad Laboratories, Hercules, CA, USA) was used to quantify the density of the target protein in the membranes.
10
Western Blot Analysis of H3K27Ac Histone
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Frozen tissue was thawed and dissociated with gentleMACS Dissociator (130-093-235, Macs Miltenyi Biotec). Protein was extracted in Tissue Extraction Reagent (FNN071, Thermo Fisher Scientific) and concentration determined with Pierce BCA Protein Assay Kit (23225, Thermo Fisher Scientific). 60 μg of protein was separated by electrophoresis in a 4–15% precast protein gel (4561086, BioRad) and transferred to PVDF membranes. Blocking was subsequently performed with 5% non-fat milk in TBST, followed by incubation with anti-H3K27Ac antibody at 1:500 dilution (8173S, Cell Signaling Technology) overnight. After 5 washes with TBST, membranes were incubated with HRP-conjugated anti-Rabbit IgG antibody at 1:1000 (7074 Cell Signaling Technology) for 1 hour. Pierce ECL Plus (32132, Thermo Fisher Scientific) was used to detect protein bands. Blots were then stripped (46430, Thermo Fisher Scientific) and re-probed with anti-total H3 primary antibody at 1:1000 dilution (14269S, Cell Signaling Technology) as a loading control. HRP-conjugated anti-Mouse IgG antibody (7076, Cell Signaling Technology) was used to detect total H3 signal. Protein from a patient-derived DIPG cell line SF8628 was extracted with RIPA buffer (89900, Thermo Fisher Scientific) and processed in parallel with tissue specimens as a positive control. Densitometry analysis was performed with image J.
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