The following were purchased from Sigma-Aldrich (Dorset, UK): Dulbecco’s Modified Eagle Medium (DMEM), Penicillin/Streptomycin, MEM non-essential amino acids 100×, 0.25% Trypsin-EDTA, L-glutamine, putrescine, spermidine, spermine. Fetal bovine serum (FBS) was from Gibco by Life Technologies (Sao Paolo, Brazil), mTOR siRNA and negative control were from Fisher Chemical (Loughborough, UK), protease and phosphatase inhibitors cocktail from Roche (Indianapolis, IN, USA), BCA protein assay kit from BioVision (Milpitas, CA, USA). RIPA lysis buffer 10× from Millipore (Burlinton, MA, USA). The following antibodies were purchased from Abcam Biotechnology (Cambridge, UK) and used at the indicated dilutions: rabbit polyclonal anti-actin antibody (ab119716, 1:7, 500 dilution), goat polyclonal anti-rabbit IgG-HRP (ab205718, 1:10,000 dilution). Additionally, mouse monoclonal anti-ODC antibody (sc-398116, 1:100 dilution), mouse monoclonal anti-phospho-4EBP1 (sc-293124, 1:200 dilution), mouse monoclonal anti-phospo-p70S6K (sc-8416, 1:300 dilution), mouse monoclonal anti- eIF4E (sc-271480, 1:300 dilution) and goat anti-mouse IgG-HRP (sc-2005, 1:2,500 dilution) antibodies were from Santa-Cruz Biotechnology (Heidelberg, Germany). Rapamycin and the class 1 dual inhibitors of PI3K and mTORC1, NVP-BEZ235, were from Cayman Chemicals (Ann Arbor, MI, USA).
Protease and phosphatase inhibitors cocktail
Manufactured by Roche
Sourced in United States, Switzerland, United Kingdom, China
Protease and phosphatase inhibitors cocktail is a laboratory product that contains a mixture of chemical compounds designed to inhibit the activity of proteases and phosphatases in biological samples. This product is used to preserve the integrity of proteins and phosphorylation states during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus, by Thermo Fisher Scientific (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Dulbecco s modified eagle medium dmem, by Merck Group (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Chemidoc, by Bio-Rad (3 mentions)
Bradford assay, by Bio-Rad (3 mentions)
Ripa lysis buffer 10, by Merck Group (2 mentions)
Goat polyclonal anti rabbit igg hrp, by Abcam (2 mentions)
Goat anti mouse igg hrp sc 2005, by Santa Cruz Biotechnology (2 mentions)
L glutamine, by Merck Group (2 mentions)
Gradient mini precast tgx gel, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Odyssey clx imaging system, by LI COR (2 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Merck Group (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx gel, by Bio-Rad (2 mentions)
Hrp conjugated secondary igg antibodies, by Cell Signaling Technology (2 mentions)
46 protocols using protease and phosphatase inhibitors cocktail
1
Regulation of mTOR Signaling Pathway
2
Protein Extraction and Western Blot Analysis
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Skin was collected from the Acetone and TPA treated wild-type and K14-sPLA2-IIA mice and flash-frozen in liquid Nitrogen. The epidermis was scraped and minced in the RIPA buffer containing protease and phosphatase inhibitors cocktail (Roche). The tissue was homogenized for 10 min and frozen at −80 °C until further use. For the preparation of the whole cell lysates of cultured cells, the cells were washed with ice-cold PBS and scraped in the RIPA buffer. Next, the cell lysate was centrifuged at 16,000 ×g for 30 min at 4 °C. Protein concentration was quantified using Bradford reagent (Sigma) as per manufacturer's instruction. Total 40–60 μg of protein was loaded on 10% SDS-polyacrylamide gel and transferred on to nitrocellulose membrane. Membrane was then blocked with blocking buffer (5% nonfat dry milk or 5% BSA, 10 mM Tris, 150 mM NaCl, 0.1% Tween-20) and probed with the following primary antibodies: p-c-Jun (1:1000), c-Jun (1:1000), p-c-Fos (1:1000), c-Fos (1:1000) all from Cell Signalling Technologies, USA, anti-sPLA2-IIA (1:500) from Merck-Millipore and β-actin (1:10000) from Sigma Aldrich.
3
Western Blot Analysis of Protein Expression
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Protein lysates were obtained by homogenization in RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) with protease and phosphatase inhibitors cocktail (Hoffmann-La Roche). Protein extracts were quantified by using the Bradford assay (Bio-Rad, Hercules, CA, USA) and equal amounts of total protein (40 μg/lane) were separated by 4–15% gradient mini precast TGX gel (Bio-Rad) before transferring to nitrocellulose by standard western conditions, blocked in BSA solution and primary antibodies (1:1000 in BSA solution; incubated overnight at 4 °C). The secondary antibody (1:3000 in 5% milk/TBS/Tween20 solution) was incubated at RT for 1 h before detection. Immunocomplexes were detected with the enhanced chemiluminescence kit ECL plus, by Thermo Fisher Scientific (Rockford, IL, USA) using the ChemiDoc (Bio-Rad). Values were normalized to α-tubulin. Each experiment was performed in duplicate.
4
Quantitative Protein Expression Analysis
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Western blots analyses for protein expression were performed on left ventricle homogenates, as described previously (37 (link)). Left ventricular tissue was homogenized with RIPA lysis buffer (Thermo Scientific) with protease and phosphatase inhibitors cocktail (Roche) and protein lysates were separated by centrifugation at 12,000 g and 4°C for 15 min. Protein concentration in tissue homogenates was determined using a Pierce BCA protein assay kit (Thermo Scientific). Equal amounts of protein (30 µg) were separated by SDS-PAGE on 4%–15% Mini-PROTEAN TGX gels (Bio-Rad) and transferred on to PVDF membrane and blocked with 1% BSA in TBS-T at room temperature for 60 min. The membranes were incubated overnight with primary antibody against TLR4 (48–2,300 Invitrogen, 1:1,000 dilution). Immunodetection was accomplished by incubation with IRDye secondary antibodies for 1 h at room temperature and imaged using Odyssey CLx imaging system (Licor). Protein expression of β-Actin or GAPDH was used as a loading control. The density of protein bands was quantitatively analyzed by Image J software (NIH) and expressed as a relative ratio against the loading control.
5
Immunoprecipitation of V5-Cfdp1 and Myc-Arp6
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HeLa cells were transfected with 2 μg of V5-Cfdp1 and Myc-Arp6 or HA-H2A.Z expression vectors by using Lipofectamine 3000 reagent according to the manufacture’s protocol, and 48 h later, they were lysated in IP buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP-40, 5 mM EGTA, 5 mM EDTA, 20 mM NaF) supplemented with protease and phosphatase inhibitors cocktail (from Roche) and 1 mM PMSF. Cleared lysates were immunoprecipitated O/N at 4 °C with 2 μg of mouse anti-V5 antibodies (Invitrogen) followed by 1 h of incubation with 30 μl of agarose-conjugated protein A/G (Santa Cruz Biotechnology). The beads were then washed three times in IP buffer and analyzed by immunoblotting.
6
Immunoprecipitation of NRP1 Protein
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Cells were washed with cold PBS and lysed in 50 mM Tris buffer (pH 7.4) containing 150 mM NaCl, 1% Triton X-100, 1 mM EDTA and protease and phosphatase inhibitors cocktail (Roche). For cytosol and membrane proteins extractions, cells were prepared using a subcellular fractionation kit (Thermo Scientific) with protease and phosphatase inhibitors according to the manufacturer’s instructions.
For immunoprecipitation, proteins were prepared using lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10% glycerol, and protease and phosphatase inhibitors. Five hundred micrograms of total protein extract was incubated with 1 µg of anti-NRP1 antibody or control IgG overnight at 4 °C. Complexes were pulled down using Bio-Adembeads Protein A/G magnetic beads (Ademtech), washed with lysis buffer and analysed by SDS-PAGE. Immunostaining was visualised using the GBox system (Syngene). Band intensities were quantified using the Multi Gauge v3.0 software (Fujifilm).
For immunoprecipitation, proteins were prepared using lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10% glycerol, and protease and phosphatase inhibitors. Five hundred micrograms of total protein extract was incubated with 1 µg of anti-NRP1 antibody or control IgG overnight at 4 °C. Complexes were pulled down using Bio-Adembeads Protein A/G magnetic beads (Ademtech), washed with lysis buffer and analysed by SDS-PAGE. Immunostaining was visualised using the GBox system (Syngene). Band intensities were quantified using the Multi Gauge v3.0 software (Fujifilm).
7
Pioglitazone Alters Protein Expression
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H1299 and H460 cancer cells were seeded into 100 mm3 petri dishes and treated for 24 h with pioglitazone [1 and 10 μM]. Protein lysates were obtained by homogenization in RIPA lysis buffer (Sigma-Aldrich, MO, USA) with protease and phosphatase inhibitors cocktail (Hoffmann-La Roche). Protein extracts were quantified by using Bradford assay (Bio-Rad, CA, USA) and equal amounts of total protein (40 μg/lane) were separate by 4–15% gradient mini precast TGX gel (Bio-Rad) before transferring to nitrocellulose by standard western conditions, blocked in BSA solution and primary antibodies (1:1000 in BSA solution; incubated overnight at 4 °C). The secondary antibody (1:3000 in 5% milk/TBS/Tween20 solution) was incubated at RT for 1 h before detection. Immunocomplexes were detected with the enhanced chemiluminescence kit ECL plus, by Thermo Fisher Scientific (Rockford,IL) using the ChemiDoc (Bio-Rad). Values were normalized to α-tubulin. Each experiment was done in duplicate.
8
Protein Extraction and Western Blotting
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Tissues that were snap frozen in liquid N2 were homogenized in RIPA buffer containing protease and phosphatase inhibitors cocktail (Roche and Thermo Fisher Scientific), protein extracted, and then subjected to Western blotting. Equal amounts (30 µg) of protein were loaded onto minigels (Bio-Rad Laboratories) and transferred onto a polyvinylidene fluoride membrane using established protocols. Samples were probed for the indicated proteins as described previously (Jin et al., 2011 (link)) using the antibodies listed in the following section.
9
Western Blot Assay Protocol
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The Western blot assay was executed as previously described [49 (link)]: 1 × 106 cells were seeded in Petri dishes (ø 6 cm, 5 mL/dish) and incubated overnight. On the next day, the cells were treated with the compounds in fresh culture media (5 mL/dish) for the indicated time. Next, cells were harvested with a cell scraper and lysed in lysis buffer including protease and phosphatase inhibitors cocktail (Roche, Mannheim, Germany). Cell debris were removed from the protein lysates via centrifugation. Afterwards, the cell-free protein mixtures were separated in gradient ready-made Mini-PROTEAN® TGX Stain-FreeTM gels (Bio-Rad, Hercules, CA, USA) using SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis). Then, the proteins were blotted onto ø 0.2 µm pore PVDF membrane. The membrane was blocked and consequently treated with primary and secondary antibodies for protein detection. The signals were detected using the ECL chemiluminescence system (Thermo Scientific, Rockford, IL, USA). The antibodies used are listed in Table 2 .
10
Quantifying SOX9 Protein Levels in TNBC
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Western blotting was used to determine the protein expression levels in TNBC cells. Total protein (40 µg) was extracted from MDA-MB-231 and MDA-MB-468 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology), supplemented with protease and phosphatase inhibitors cocktail (Roche Diagnostics). The protein concentration was detected using a BCA Protein Assay kit. A total of 40 µg of protein was loaded and separated via 12% SDS-PAGE, followed by transfer to PVDF membranes (EMD Millipore). Membranes were blocked with 5% skimmed milk dissolved in PBS-Tween (0.1% Tween) at room temperature for 1.5 h and incubated with primary antibodies overnight at 4°C. After incubating with peroxidase-conjugated secondary antibodies for 2 h at room temperature, the protein abundance was analyzed using an enhanced chemiluminescence system (EMD Millipore). Primary antibodies against SOX9 (1:1,000; cat. no. ab185966) and GAPDH (1:1,000; cat. no. ab8245) were purchased from Abcam. HRP-conjugated anti-mouse (1:5,000; cat. no. sc-2005) or anti-rabbit (1:5,000; cat. no. sc-2004) secondary antibodies were from Santa Cruz Biotechnology, Inc.
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