Treated cells were harvested in radioimmunoprecipitation assay buffer (RIPA; Beyotime, P0013B) supplemented with protease inhibitor (Roche, 04693159001) and phenylmethylsulfonyl fluoride (PMSF; Beyotime, ST506) on ice, and the protein concentration was determined by using a BCA Protein Assay Kit (Thermo Scientific, 23250). Cellular proteins were separated via SDS-PAGE through 8–12% gels before they were transferred to polyvinylidene fluoride (PVDF) microporous membranes (EMD Millipore, IPVH00010). The blots were probed for 12 h at 4 °C with primary antibodies, and secondary antibodies were incubated with the PVDF membrane for 1 h at room temperature. Visualization was performed with a Molecular Imaging System (Carestream Health, INC., NY, USA). The antibodies used are as follows: anti-SLC40A1 rabbit antibody (Abcam, ab78066), anti-GCLM rabbit antibody (HuaBio, ET1705-87), anti-GCLC rabbit antibody (HuaBio, ET1704-38), anti-IκBα rabbit antibody (HuaBio, ET1603-6), anti-phospho-IκBα rabbit antibody (HuaBio, ET1609-78), anti-NF-kB p65 rabbit antibody (HuaBio, ET1603-12), anti-SLC7A11 rabbit antibody (Abcam, ab175186), anti-c-Jun rabbit polyclonal antibody (Proteintech, 24909-1-AP), anti-ferritin rabbit monoclonal antibody (Abcam, ab75973), anti-GAPDH rabbit antibody (HuaBio, R1210-1), and anti-β-actin antibody (HuaBio, 1102-1).
Ipvh00010
Manufactured by Merck Group
Sourced in United States, Germany, China, Ireland, United Kingdom, France, Canada
IPVH00010 is a laboratory equipment product manufactured by Merck Group. It is designed for specific laboratory applications. The core function of this product is to assist in various scientific and research activities. However, a detailed description of its features and intended use cannot be provided while maintaining an unbiased and factual approach.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (47 mentions)
Ripa lysis buffer, by Beyotime (45 mentions)
Wbkls0500, by Merck Group (27 mentions)
Bca protein assay kit, by Beyotime (27 mentions)
Protease inhibitor cocktail, by Merck Group (23 mentions)
Ripa buffer, by Beyotime (21 mentions)
Protease inhibitor cocktail, by Roche (20 mentions)
Chemidoc mp imaging system, by Bio-Rad (20 mentions)
Las 4000, by Cytiva (19 mentions)
Ripa buffer, by Merck Group (18 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (18 mentions)
Ab8245, by Abcam (16 mentions)
β actin, by Merck Group (15 mentions)
Amersham imager, by Cytiva (15 mentions)
Wbkls0100, by Merck Group (15 mentions)
Bca kit, by Thermo Fisher Scientific (15 mentions)
P0013, by Beyotime (15 mentions)
Ripa lysis buffer, by Solarbio (14 mentions)
Image lab software, by Bio-Rad (13 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (13 mentions)
955 protocols using ipvh00010
1
Immunoblotting Assay for Cellular Protein Analysis
2
Western Blot Analysis of Aortic Proteins
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Protein levels of genes in the aorta were detected by Western blotting analysis. In brief, aortic tissue lysates were separated by 10% SDS-PAGE and transferred to 0.45 μm PVDF membranes (IPVH00010, Merck Millipore). After blocking in 5% skim milk (Cat#D8340, Solarbio; for non-phosphorylated protein) or 5% BSA (G5001-5G, Servicebio; for phosphorylated protein) for 2 h, the membranes were incubated overnight at 4°C with primary antibodies: p65NF-κB (1:1000, AF5006, Affinity Biosciences); p-p65NF-κB (1:1000, AB76302, Abcam); p38 mitogen-activated protein kinase (p38MAPK; 1:2500, ab170099, Abcam); p-p38MAPK (1:1000, AB195049, Abcam); VCAM-1 (1:5000, ab134047, Abcam); and β-actin (1:1000, GB15003, Servicebio). The membranes were then incubated with HRP-conjugated secondary antibody for 90 min at room temperature. Protein bands were visualized using ECL solution on Ultra Sensitive Multifunctional Imager (AI680RGB, GE, Japan) and analyzed using the ImageJ software.
3
Profiling Epigenetic Regulators in Cells
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Cells were lysed in ice-cold RIPA cell buffer (P0013B, Beyotime Biotechnology) supplemented with PMSF (ST507, Beyotime Biotechnology). The protein samples were then separated via SDS‒PAGE (C671102, Sangon Biotech) and electrotransferred to a PVDF membrane (IPVH00010, Merck Millipore). Subsequently, the PVDF membrane was blocked with 5% skim milk for 4 h. After blocking, the membrane was incubated with specific primary antibodies overnight at 4 °C and followed by incubation with an HRP-conjugated secondary antibody at room temperature for 1 h. Images were acquired with a Tanon-5200Multi chemiluminescence gel imaging system (Tanon). The primary antibodies used in this study are FLAG (SG110–26, GNI 1:1000), Prdm14(D121722, BBI, 1:1000), Dnmt1(381634, ZENBIO, 1:1000), Dnmt3a(R26690, ZENBIO, 1:1000), Dnmt3b(UPA62506, Gene Universal, 1:1000), Dnmt3l (UPA05797, Gene Universal, 1:1000), PKCα(R25382, ZENBIO, 1:1000), PKCβ(R25383, ZENBIO, 1:1000), PKCδ(R25384, ZENBIO, 1:1000), β-tubulin (200608, ZENBIO, 1:2000), Suv39h1(10574-1-AP, Proteintech, 1:1000), Suv39h2(11338-1-AP, Proteintech, 1:1000), H3K9me2(39239, Active Motif, 1:1000), H3K9me3(39161, Active Motif, 1:1000), H3K27me3 (39157, Active Motif, 1:1000), Ezh2(21800-1-AP, Proteintech, 1:1000), and Ezh1(20852-1-AP, Proteintech, 1:1000).
4
Western Blot Analysis of hBMSCs
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Transfected hBMSCs were lysed with radio immunoprecipitation buffer (Beyotime Institute of Biotechnology) and a Bradford kit (Beyotime Institute of Biotechnology) was used to determine total protein concentration. Proteins (20 µg/lane) were isolated by SDS-PAGE on 10% gels and were electrotransferred onto nitrocellulose membranes (cat. no. IPVH00010; EMD Millipore); membranes were then blocked with 1% BSA (Beyotime Institute of Biotechnology) to ensure non-specific binding for 60 min at room temperature. Membranes were incubated with anti-rabbit alkaline phosphatase (ALP; 1:1,000; cat. no. ab75699; Abcam), anti-rabbit RUNX2 (1:1,000; cat. no. ab192256; Abcam) and anti-rabbit GAPDH (1:20,000; cat. no. ab181602; Abcam) primary antibodies at 4°C overnight. Anti-HRP (1:2,000; cat. no. ab181658; Abcam) secondary antibody was used to incubate the membrane for 1 h at room temperature. All antibodies were diluted with dH2O (Takara Bio, Inc.). Protein bands were observed by enhanced chemiluminescence (Beyotime Institute of Biotechnology) and exposed on X-ray film (Kodak). The relative grey values were analyzed by ImageJ 1.48 software (National Institutes of Health).
5
TGF-β3-Induced SMAD2 Phosphorylation Assay
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Approximately 2.5 × 105 of MC38 and KPC1 cells were plated in 6-well plate in complete medium and incubated overnight at 37 °C. The next day, the complete medium was replaced with 0.2% FBS medium and further incubated at 37 °C for eight hours. Cells were then treated with 1 µg/mL of LY364947 for 30 min before stimulating with 5 ng/mL of TGF-β3 for 2 h. Cells were lysed in radioimmunoprecipitation assay buffer (RIPA) sampler buffer (50 mM Tris–HCl (pH 8.0) with 150 mM NaCl, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) containing cOmplete™ Protease Inhibitor Cocktail (11697498001, Roche, Basel, Switzerland). Protein concentration was determined using a DC™ Protein Assay Kit (5000111, Bio-Rad, Hercules, CA, USA). An equal amount of protein was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane (IPVH00010, Merck Millipore). Membrane was probed with phospho-SMAD2 antibody [40 (link)] (homemade) and GAPDH antibody (AB2302, Merck Millipore, Billerica, MA, USA). The chemiluminescent signal was detected using the Clarity™ Western ECL Substrate (Hercules, CA, USA) and visualized using the ChemiDoc™ Imaging Systems (17001402, Bio-Rad, Hercules, CA, USA).
6
Western Blot Analysis of Protein Targets
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Cells were lysed in lysis buffer (Beyotime, P0013) with 1 mM PMSF and protease inhibitors (Roche, 4693116001). After quantification of protein using a protein assay kit (Thermo Fisher Scientific, 23225), equivalent protein were separated by SDS-PAGE and then transferred to PVDF membranes (Merck Millipore, IPVH00010). Membranes were blocked in TBST with 5% skim milk for 1 h at room temperature. Target proteins were detected by overnight incubating with the indicated primary antibodies at 4 °C, followed by the corresponding HRP-conjugated secondary antibodies at room temperature for 2 h. Immunoreactivity was detected with FDbio-Dura ECL kit (Fudebio-tech, FD8020). Membranes were imaged by an automatic chemiluminescence image analysis system (Tanon Science & Technology, Shanghai, Tanon 5200). Band intensity was quantified by ImageJ software.
7
Western Blot Protein Quantification
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The protein samples were loaded 20μg per sample for SDS-PAGE, and then transferred to the PVDF membrane (IPVH00010, Merck KGaA, Darmstadt, Germany). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with the primary antibodies (shown in Supplementary Methods ) at 4°C overnight and then secondary antibodies (shown in Supplementary Methods ). The target protein bands were quantified by Fusion Solo system (Vilber, France) and measured with software (Image J software, Research Services Branch, National Institute of Mental Health, Bethesda, MD, USA) for statistical analyses.
8
Immunoblotting Analysis of BMSCs under IL-17 and TNFα
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For immunoblotting analysis, BMSCs incubated with 50 ng ml–1 IL-17 for 15, 30, and 60 min or 10 ng ml–1 TNFα for 7.5, 15, 30 min were pre-challenged by 100 nM RA for 6 h. Cells were washed with ice-cold PBS, harvested and lysed for 15 min by lysis buffer containing 0.5% TritonX-100 (T9284, Sigma), 20 mM Hepes pH7.4 (H-4034, Sigma), 150 mM NaCl (A100241, Sangon Biotech), 12.5 mM β-glycerophosphate (A500486, Sangon Biotech), 1.5 mM MgCl2 (M4880, Sigma), 2 mM EGTA (A600077, Sangon Biotech), and a cocktail of protease inhibitors, Na3VO4 (A600869, Sangon Biotech), NaF (A500850, Sangon Biotech), and PMSF (A610425, Sangon Biotech). Equal amounts of protein extracts were resolved in 10% SDS-PAGE and transferred to PVDF membranes (IPVH00010, Merck Millipore). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST, pH 7.6) for 1 h at room temperature before incubated overnight with the primary antibodies (p65 1:1,000, pp65 (Ser536) 1:1,000, IκBα 1:1,000, pIκBα (Ser32) 1:1,000, β-actin 1:1,000) at 4°C and then incubated with the secondary antibodies (rabbit, 1:10,000, W401B, Promega) for 1 h at room temperature. Finally, the blots were detected by enhanced chemiluminescent reagents (Millipore).
9
Autophagy Regulation by PIONs in U251 Cells
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After incubation with various concentrations of PIONs@ E6 for 24 hours, U251 cells were homogenized in lysis buffer, separated by 10% SDS-PAGE, and transferred to PVDF membranes (IPVH00010, EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% skimmed milk and then incubated with diluted primary antibodies including rabbit anti-P62 (1:10000; ab109012, Abcam, MA, USA), anti-beclin-1(1:2000; ab207612, Abcam), anti-LC3II (1:2000; ab192890, Abcam), anti-LC3I (1:2000; ab192890, Abcam), and anti-GAPDH (1:1000; ab181602, Abcam). Blots were washed with TBS/TWEEN and incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (1:5000; KGAA35, Keygen Inc., Nanjing, P.R.C) for 2 hours. After washing with TBS/TWEEN, the blots were developed with the chemiluminescence method (ECL Luminata Crescendo, WBLUR0500, EMD Millipore).
10
Protein Extraction and Immunoblotting of Cell-Derived EVs
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A confluent layer of transfected U87 cells, conditioned medium and derived-EV pellets were washed once with ice-cold PBS and lysed into seven volumes of Lysis buffer (25 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1% NP-40), then lysed on ice for 1 h, and the samples were then centrifuged at 22,000xg for 30 min at 4 °C. The supernatants were collected, and the total protein content was calculated using the BCA Protein Assay Kit (71,285-M, www.thermofisher.com ). Ten micrograms of protein samples were mixed with 2X Laemmli Sample Buffer (1,610,737, www.Bio-Rad.com ) added with 50 mM dithiothreitol, heated to 37 °C for 10 min, resolved in a 13% polyacrylamide gel, and transferred onto PVDF membranes (IPVH00010, www.merckmillipore ) for immunoblot analysis.
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