Whole heart tissue and cultured cells were homogenized in lysis buffer containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich; P8340, P5726, P2850). Bradford assay was performed to analyze and normalize protein concentrations and lysates were prepared by addition of NuPAGE LDS Sample Buffer (ThermoFisher; NP0007) and 100 µM dithiothreitol (Bio-Rad; 161–0611). Samples were sonicated and boiled for 5 min at 95 °C and stored at −80 °C. Proteins were separated on a 4–12% NuPAGE Bis–Tris gel (ThermoFisher; NP0321BOX) and transferred onto a polyvinylidene fluoride membrane. Nonspecific binding sites were blocked using Odyssey blocking buffer (LICOR, 927–60001) and proteins were labeled with primary antibodies in 0.2% Tween in Odyssey blocking buffer overnight. After multiple washes, blots were incubated with secondary antibodies in 0.2% Tween 20 in Odyssey blocking buffer for 1.5 h at room temperature and scanned using the LICOR Odyssey CLx scanner. Quantification was performed using ImageJ software. Antibodies and their dilutions are listed in Supplementary Table 1 .
P2850
Manufactured by Merck Group
Sourced in United States, Germany
The P2850 is a lab equipment product manufactured by the Merck Group. It is designed to perform a core function within a laboratory setting, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further information is not available.
Automatically generated - may contain errors
Lab products found in correlation
P8340, by Merck Group (17 mentions)
P5726, by Merck Group (12 mentions)
Ripa buffer, by Merck Group (4 mentions)
Dithiothreitol (dtt), by Bio-Rad (2 mentions)
Odyssey clx scanner, by LI COR (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
S7653, by Merck Group (2 mentions)
S7920, by Merck Group (2 mentions)
E3889, by Merck Group (2 mentions)
G5516, by Merck Group (2 mentions)
Res3098t b7, by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Complete mini, by Merck Group (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Np0321box, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Cell Biolabs (1 mentions)
Hrp conjugated secondary antibody, by Merck Group (1 mentions)
Femto ecl, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
35 protocols using p2850
1
Western Blot Protein Analysis Protocol
2
Protein Extraction from Myocardial Tissue
3
Protein Expression Quantification Protocol
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Cells were grown in 6-well plate (2.5 x 105 cells/well) and infected the following day with 5000 pp/cell using the synchronous infection protocol as described above. At a given time point, cells were washed with PBS (Gibco, 70013–016) and harvested using PBS with 0.6 mM EDTA followed by a centrifugation for 5 min at 5000 g at 4°C. Cell pellets were lysed in 160 μL of lysis buffer (25 mM Tris pH7.4, 150 mM NaCl; 1 mM EDTA pH8; 5% glycerol; 1% NP40; 1 mM PMSF) supplemented with phosphatase inhibitors 1 and 2 (Sigma, P2850 and P5726). Clarified lysates were denatured in loading buffer (50 mM Tris pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 0.05% bromophenol blue) and separated by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on 0.2 μm nitrocellulose membrane (GE Healthcare, 10600001). Membranes were blocked for 30 min in saturation buffer (5% BSA, 0.1% Tween20 in TBS) followed by overnight incubation with primary antibodies (see Table 3
4
Protein Extraction and Western Blot Analysis
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For protein extraction, cells were washed twice with ice‐cold PBS and lysed on ice in golden lysis buffer (20 mmol/L Tris‐HCl (Sigma Aldrich RES3098T‐B7, St. Louis, MI), pH 8.0, 137 mmol/L NaCl (Sigma Aldrich S7653, St. Louis, MI), 5.95 mmol/L EDTA (Sigma Aldrich 1233508, St. Louis, MI), 5 mmol/L EGTA (Sigma Aldrich E3889, St. Louis, MI), 10 mmol/L NaF (Sigma Aldrich S7920, St. Louis, MI), 1% Triton X‐100 (Sigma Aldrich X100, St. Louis, MI), and 10% glycerol (Sigma Aldrich G5516, St. Louis, MI)) supplemented with protease inhibitors (Sigma Aldrich P8340, St. Louis, MI) and phosphatase inhibitors (Sigma Aldrich P2850, St. Louis, MI). The proteins were separated via 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to PVDF membranes (Thermo Fisher Scientific LC2002, CA). HAS3 (Sigma Aldrich SAB2108148, St. Louis, MI), GFAP (GeneTex GTX108711, CA), β‐tubulin (GeneTex GTX11307, CA), TUBB3 (GeneTex GTX631836, CA) and α‐actin (GeneTex GTX109639, CA) antibodies were diluted 1:2000 in TBST, and the membranes were incubated for 2 hours at room temperature. Horseradish peroxidase (HRP)‐conjugated anti‐mouse and anti‐rabbit IgG (Santa Cruz Biotechnology SC‐2005, CA) secondary antibodies were diluted 1:4000, and the membranes were incubated for 1 hour at room temperature. α‐Actin was used as the protein loading control.
5
Nuclear Protein Extraction Protocol
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For nuclear protein extraction cells were trypsinized (0.025% trypsin and 0.04% EDTA, SIGMA 25300-054) at 37 °C, pelleted at 300 rcf and washed twice with cold PBS. During each wash cells were pelleted at 300rcf for 5 min at 4 °C. Cells were incubated in hypotonic buffer (10 mM Tris-HCl pH 7.8, 5 mM KCl, 2 mM MgCl2 DTT 1 mM) containing protease inhibitors (SIGMA P8340) for 10 min at 4 °C. Cells were pelleted at 300 rcf for 5 min at 4 °C and plasma membrane lysis was performed in 0,25% NP-40 hypotonic buffer on ice for 15 min. Nuclei were pelleted at 300 rcf for 15 min at 4 °C and washed twice in hypotonic buffer. Isolated nuclei were incubated in RIPA buffer (150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris-HCl, pH 8.0) containing protease (SIGMA P8340) and phosphatase inhibitors (SIGMA P2850). Insoluble material was pelleted by centrifugation at 16,000 rcf for 30 min at 4 °C. Protein concentrations were determined using the Bradford assay (Bio-Rad 500-0006). Western blot was performed as specified in the apposite section with the antibodies indicated in Table S6 .
6
Immunoblotting of Phosphorylated ERK1/2
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STHdh cells were washed with PBS, harvested, and lysed using RIPA buffer (R0278; Sigma) with protease (P8340; Sigma) and phosphatase inhibitor cocktails (P2850 and P5726; Sigma). Lysates were solubilized in sodium dodecyl sulphate (SDS) sample buffer, separated on 10% SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; EMD Millipore Corporation, Billerica, MA). Transfer membranes were blocked for 30 min at room temperature with Blocking One-P (Nakalai Tesque, Kyoto, Japan), then incubated overnight at 4°C with the primary antibodies (rabbit anti-phospho-extracellular signal-regulated kinase 1/2 [ERK1/2] [Cell Signaling Technology, Danvers, MA], rabbit anti-total-ERK1/2 [Cell Signaling Technology], and mouse anti-β-actin [Sigma]). Subsequently, the membranes were incubated with secondary antibodies (peroxidase-conjugated goat anti-rabbit IgG [Thermo Fisher Scientific Inc., Franklin, MA] or peroxidase-conjugated goat anti-mouse antibody [Thermo]). The immunoreactive bands were visualized using ImmnoStar®LD (Wako Pure Chemical Industries, Osaka, Japan) and the LAS-4000 Luminescent Image Analyzer (Fuji Film Co., Ltd., Tokyo, Japan).
7
Tissue Homogenization and Protein Extraction
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The WAT was homogenised by grinding with a cold mortar and pestle into ice-cold RIPA buffer (Cell Signaling Technologies, 9806; Danvers, MA, USA) containing protease and phosphatase inhibitors 1 and 3 (Sigma-Aldrich, P8340, P2850, and P0044; St. Louis, MO, USA). The tissue homogenates were kept on ice for 30 min before freeze-thawing 2 times using liquid nitrogen to assist the tissue lysis. The tissue debris was removed by centrifugation for 30 min at 12,000 rpm, 4 °C, and the cleared lysates were transferred to new tubes, avoiding the floating lipid layer. The total protein levels were quantitated by a DC protein assay (Bio-Rad 5000112; Hercules, CA, USA).
8
Protein Extraction and Quantification from Frozen Tissues
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Total protein from frozen liver tissue or cell lines was extracted in RIPA lysis buffer (1.6 mM Na2HPO4, 8.4 mM NaH2PO4, 0.1 M NaCl, 0.1% SDS, 0.1% Triton X-100) supplemented with 10 mM sodium deoxycholate, 1 mM sodium orthovanadate, 50 mM NaF, protease (P8340, Sigma-Aldrich) and phosphatase inhibitor cocktails (P2850, Sigma-Aldrich), in addition to 10 mM N-ethylmaleimide (NEM) and 10 mM iodoacetamide (IAA) cysteine protease inhibitors, which prevent non-specific deSUMOylation. For tissue homogenization, two cycles of 5,000 rpm for 30s in the Precellys 24 homogenizer (Bertin Instruments) were performed. Lysates were clarified by centrifugation (12,500 rpm, 20 min, 4°C) and total protein concentration from the supernatant was estimated by the Micro BCA Protein Assay Kit (23235, Thermo Fisher Scientific) using a BSA standard curve, in a SpectraMax M2/M2e microplate reader (Molecular Devices). For western blotting analysis, 10–25 μg of total protein were combined with 5x Laemmli sample loading buffer (250 mM Tris pH 6.8, 10% SDS, 50% glycerol, 500 mM β-mercaptoethanol, bromophenol blue).
9
Protein Extraction from Embryo and Fin Tissues
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Freshly extracted samples were placed into 50 µl RIPA buffer (R0278, sigma-aldrich, MO, USA) containing 1:1,000 phosphatase inhibitor cocktails 1 and 2 (P2850, P5726, sigma-aldrich) and 1X protease inhibitor cocktail (cOmplete Mini, EDTA-free, sigma-aldrich). Samples were homogenised using a hand-held motorised homogeniser with individual sterile plastic pestles (PELLET PESTLE cordless motor, 749540-0000, and pestles, 749521-1500, Kimble Chase, DWK Life Sciences LLC, Wertheim/Main, Germany), then debris pelleted by centrifugation at > 12,000×g for 10 min at 4 °C. 37.5 µl supernatant containing protein was transferred into a new Eppendorf tube containing 12.5 µl 4 × SDS Sample Buffer (novagen, EMD Millipore Corp, MA, USA). Samples were denatured by boiling at 95 °C for 5 min and stored at − 20 °C. Embryo samples were typically pooled, with ~ 2–3 embryos yielding 1 sample, while individual fin tissue samples yielded sufficient protein.
10
Protein Extraction from Cell Pellets
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Cell pellet was lysed in 3–5 volumes of RIPA buffer (R0278, Sigma-Aldrich, St. Louis, MO, USA) supplemented with protease (#5871, Cell Signaling Technology, Danvers, MA, USA) and phosphatase inhibitors (P2850, Sigma-Aldrich). Following 30 min of incubation on ice, cell lysate was spun down (14,000× g for 15 min at 4 °C) and the supernatant was recovered. Finally, the protein concentration of each sample was defined by the Bradford method (39222, Serva, Heidelberg, Germany).
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