Protein samples were extracted using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). 20–40 μg of the cell lysate, which were determined by the BCA protein assay, were separated on 12% SDS-PAGE gels and electrophoretically transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). After blocking, the membranes were incubated overnight at 4°C with primary antibodies to CD32, CD64 (R&D Systems, Minneapolis, MN,USA), p38, phospho-p38(Cell Signaling, US), IκBα, phospho-IκBα, phospho-IKK, phospho-p65, p65, and TWIST (Cell Signaling Technology, Danvers, MA,USA), and house-keeping protein controls such as β-actin, α-tubulin or laminin B1(BD Pharmingen, San Diego, CA, USA). After washing, the membranes were incubated with HRP-conjugated secondary antibodies (R&D Systems) for 1 h and detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, USA) reaction. Positive immunoreactive bands were quantified and normalized by β-actin using κκ The Image G program (NIH).
Hrp conjugated secondary antibody
Manufactured by R&D Systems
Sourced in United States, United Kingdom
HRP-conjugated secondary antibodies are laboratory reagents that consist of a secondary antibody covalently linked to the enzyme horseradish peroxidase (HRP). These antibodies are used to detect and visualize target proteins in various immunoassays, such as Western blotting and enzyme-linked immunosorbent assay (ELISA).
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (4 mentions)
Actin expression, by BD (4 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Gapdh, by OriGene (3 mentions)
Super signal west pico enhanced chemiluminescence system, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Ecl plus kit, by GE Healthcare (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Np40 cell lysis buffer, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor, by Merck Group (2 mentions)
Superblock t20, by Thermo Fisher Scientific (2 mentions)
Superblock tbs blocking buffer, by Thermo Fisher Scientific (2 mentions)
Tissue tek oct compound, by Sakura Finetek (2 mentions)
Fluorescent labeled secondary antibody, by Thermo Fisher Scientific (2 mentions)
Anti ki67 antibody, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
G6pd antibody, by Abcam (2 mentions)
Axio z2, by Leica (2 mentions)
Gpx4 antibody, by R&D Systems (2 mentions)
27 protocols using hrp conjugated secondary antibody
1
Western Blot Analysis of Immune Signaling
2
Western Blot Analysis of Protein Samples
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Prepped cell lysates were separated by SDS-PAGE using 10% or 12.5% gels. Protein sizes were estimated using the PrecisionPlus All Blue protein marker (Bio-Rad, USA). Gels were incubated in Bjerrum Schafer-Nielsen transfer buffer and proteins were transferred to water-activated nitrocellulose membranes (Amersham Protran Premium 0.45 NC, GE Healthcare Life Sciences, Sunnyvale, USA) using semi-dry transfer (Trans-Blot® Turbo™, Bio- Rad, USA). Next, the membranes were stained in Ponceau solution for 5 min and scanned to be used as a loading control. The membranes were blocked in 5%-10% skim milk in TBS-T (Fluka, Taufkirchen, Germany) for 1 h at RT. After blocking, the membranes were incubated with the respective primary antibodies diluted in 1% Bovine Serum Albumin (BSA) or 5% skim milk overnight at 4 °C with gentle agitation. Next, the membranes were rinsed 3 × 5 min with TBS-T, incubated 1 h at RT with HRP-conjugated secondary antibodies (R&D Systems) followed by rinsing 3 × 5 min with TBS-T. Chemiluminescence was detected using an ECLplus kit (GE Healthcare, Amersham™) and an Azure c500 system (Azure Biosystems, Dublin, USA). Protein band quantification was carried out using ImageJ software (NIH, http://rsb.info.nih.gov/ij/ ). All antibodies used are listed in Supplementary Table 5 .
3
Protein Isolation and Western Blot Analysis
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Proteins were isolated from renal tissues or MMT cells with a RIPA lysis buffer and then analyzed by Western blot. α-SMA (ET1607-53; HuaBio), Col-1 (BA0325; Bosterbio, United States), FN (BA1772; Bosterbio), FABP4 (ET1703-98; HuaBio), Saa1 (ab199030; Abcam), and GAPDH (TB802519, Origene, United States) were used as primary antibodies and the membranes were incubated with HRP-conjugated secondary antibodies (R&D Systems, United States) for more than 1 h at room temperature.
4
Immunoblotting Antibody Panel Optimization
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Anti-ARF1 and anti-ARF6 were from Proteintech (Chicago, IL). Anti-pan-actin, anti-phospho-AKT, anti-AKT, anti- pro-caspase 9, anti-pro-caspase 3, anti-EGFR, anti-phospho-ERK and anti-pro-MMP2 were all from Cell Signaling Technology (Danvers, MA). anti-ARF6, Anti-ERK, anti-FAK, anti-snail, anti-slug and anti-p-tyrosine were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-HA was from Abcam (Cambridge, MA). Anti-TurboGFP was from Thermo Fisher Scientific (Rockford, IL) and anti-Ki-67 was from BD Bioscience (Bedford, MA). HRP-conjugated secondary antibodies were from R & D Systems (Minneapolis, MN).
5
TNF-α Signaling Pathway Activation
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Cells were lysed using a RIPA lysis buffer (Beyotime, Shanghai, China) after treated with TNF-α (10 ng/ml) for 0.5,1 and 2 h. Total protein (30 μg) from each sample was subjected to the SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA), which were blocked for 2 h at room temperature with 5% nonfat milk in PBST, and then the membranes were incubated with primary antibodies at 4 °C overnight. The following antibodies were used to detect bands on the protein blots: anti-β-actin (1:1000, Abcam), anti-ERK 1/2 (1:500, Abcam), anti-phospho-ERK 1/2 (1:500, Abcam). Then the membranes were incubated with HRP-conjugated secondary antibodies (1:1000, R&D Systems, Minneapolis, MN, USA) for 2 h at room temperature. Immunoreactive material was visualized using the ImmunStar WesternC Kit (Bio-Rad, Hercules, CA, USA) products and bands were detected via ImageQuantLAS4000 (GE, Fairfield, CT, USA). All western bolt analyses were performed at least three independent experiments.
6
Evaluation of Combination Targeted Therapies
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NVP-AEW541 and NVP-BEZ235 were gifts from Novartis (Basel, Switzerland). Lapatinib, KU0063794 and LY294002 were from Cayman Chemical (Michigan, USA). Cisplatin was purchased from Sigma-Aldrich (Steinheim, Germany). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Serva (Heidelberg, Germany). Propidium iodide was from PromoCell (Heidelberg, Germany). Roswell Park Memorial Institute (RPMI) media 1640, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin [10,000 U/ml; 10 mg/ml] and trypsin-EDTA (0.05% trypsin, 0.02% EDTA in PBS) were purchased from PAN Biotech (Aidenbach, Germany). Primary antibodies were purchased from R&D Systems (Wiesbaden, Germany) (pIGF1R, IGF1R, p-EGFR, EGFR, p-ErbB2, ErbB2, p-ErbB3, ErbB3) or Santa Cruz Biotechnology (Heidelberg, Germany) (p-Akt, Akt, β-Actin, PARP). HRP-conjugated secondary antibodies were from R&D Systems. All other reagents and chemicals were from VWR BDH PROLABO (Darmstadt, Germany).
7
Western Blot Analysis of NF-κB p65 and TLR4
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Proteins were isolated from renal tissues or HK-2 cells with a RIPA lysis buffer and then analyzed by Western blotting. In brief, equal amounts of protein were separated by SDS-PAGE and then transferred on to a PVDF membrane (Bio-Rad, Hercules, CA, USA). Subsequently, the membranes were incubated with primary antibodies against NF-κB p65 (Abcam, Cambridge, MA, USA), TLR4 (Abcam, Cambridge, MA, USA), and GAPDH (Origene, Rockville, MD, USA) overnight at 4 °C. Then the membranes were incubated with HRP-conjugated secondary antibodies (R&D Systems, Minneapolis, MN, USA) for 1 h at room temperature. Finally, the proteins on the membrane were developed with an enhanced chemiluminescence agent (Millipore Corporation, Boston, MA, USA). The signals were detected using an Odyssey Infrared Imaging System (Bio-Rad, ChemiDoc MP, mANUSC, Bio-Rad Laboratories Inc., Hercules, CA, USA) and analyzed with the Image J program (National Institutes of Health, Bethesda, MD, USA). The ratio for the protein was normalized against GAPDH.
8
Immunoblot Analysis of Cleaved Caspase-3
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Basically, the analysis was performed using methods previously reported by our group23 (link). Proteins were extracted from samples using NP-40 Cell Lysis Buffer (Thermo Fisher Scientific, MA, USA) supplemented with Complete protease inhibitor (Sigma Aldrich) and Halt Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Equal amounts of protein were separated by SDS-PAGE (12.5%), transferred onto a nitrocellulose membrane, and blocked with SuperBlock T-20 (PBS; ThermoFisher Scientific). Primary antibody of cleaved caspase-3 (Cell Signaling Technology, Inc, MA, USA, #9661) were diluted to 1:1000 in SuperBlock T-20 (PBS) and incubated with blots overnight at 4 ℃. After washing in T-PBS, blots were probed with HRP-conjugated secondary antibodies (R&D Systems, Inc., MN, USA), washed in T-PBS, and then developed using the Super-Signal West Pico enhanced chemiluminescence system (ThermoFisher Scientific). Protein expression levels were normalized to the actin expression (BD Transduction Laboratories, KY, USA).
9
Detecting Pro-IL-1β and Processed IL-1β
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Pro-IL-1β and processed IL-1β forms were detected by immunoblotting, as previously described [17 (link), 50 (link), 54 (link)]. Briefly, supernatants were precipitated with 10% trichloroacetic acid (Sigma-Aldrich) and washed in cold acetone by centrifugation at 12,000 × g for 10 min at 4°C. Next, pellets were dried and resuspended in sample buffer. For analysis of cell lysates, the plates were incubated on ice for 10 min, and then the cells were detached with a scraper and transferred into 1.5-ml Eppendorf tubes. Cells were lysed with 50 μl of lysis buffer (50 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1% Triton X-100, and 5% glycerol, all purchased from Sigma-Aldrich) supplemented with a mixture of protease inhibitors (Roche). The lysates were centrifuged at 12,000 × g for 10 min at 4°C, to eliminate cellular debris and proteins were separated on 15% polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Immunoblotting was performed using goat anti-IL-1β (1:800; R&D Systems). Immunoblots were developed using HRP-conjugated secondary antibodies (1:15,000 R&D Systems) and developed with Luminata Forte Western HRP substrate (Biorad).
10
Western Blot Analysis of Cell Cycle Proteins
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Total proteins were extracted from samples using NP-40 Cell Lysis buffer containing protease inhibitor and phosphatase inhibitor. Equal amounts of protein were resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (12.5%), blotted to a nitrocellulose membrane and blocked in SuperBlock™ (TBS) Blocking Buffer (ThermoFisher Scientific, Carlsbad, CA, USA). Blots were incubated overnight at 4 °C with primary antibodies of Cyclin B1 (Abcam, Cambridge, UK), Cyclin D1 (Abcam) or PARP (Cell Signaling Technologies, Danvers, MA, USA). The blots were washed in TBS containing 0.05% Tween 20 (T-TBS), incubated with HRP-conjugated secondary antibodies (R&D Systems, Minneapolis, MN, USA), washed in T-TBS, and then developed using either the Super-Signal West Pico or the femto enhanced chemiluminescence system (ThermoFisher Scientific). The averaged protein expression was normalized to the actin expression (BD Transduction Laboratories, Lexington, KY, USA). Detailed information can be found at Supplementary Figure S2 .
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