The GR ChIP was performed as previously described [32 (link)]. We added 1 mM AEBSF or 0.1 mM PMSF, 5 mM Na+-Butyrate (NaBut), and PhosSTOP phosphatase inhibitor cocktail tablets (1 per 10 ml; Roche, Burgess Hill, UK) to all solutions unless otherwise stated. Briefly, hypothalamus tissues from four mice were cross-linked for 10 min in 1% formaldehyde in PBS. Crosslinking was terminated by adding glycine (5 min, final concentration 200 µM) and centrifuged (5 min, 6000 g, 4 °C). Pellets were washed three times with ice-cold PBS. Next, the pellets were re-suspended in ice-cold Lysis Buffer [50 mM Tris-HCl pH 8, 150 mM NaCl, 5 mM EDTA pH 8.0, 0.5% v/v Igepal, 0.5% Na-deoxycholate, 1% SDS, 5 mM NaBut, 2 mM AEBSF, 1 mM Na3VO4, complete ultra EDTA-free protease inhibitor tablets and PhosSTOP phosphatase inhibitor cocktail tablet (both 1 per 10 ml, Roche, Burgess Hill, UK)] and rotated for 15 min at 4 °C. Samples were aliquoted, sonicated (high power; 2 × 10 cycles; 30 s ON, 60 s OFF) using a water-cooled (4 °C) Bioruptor Pico (Diagenode, Liège, Belgium) and centrifuged (10 min, 20,000 g, 4 °C). Supernatants (containing the sheared chromatin) were recombined and re-aliquoted into fresh tubes for subsequent ChIP analysis and for assessment of Input DNA (i.e., the starting material). Chromatin was sonicated to a length of ~500 base pairs.
Phosstop phosphatase inhibitor cocktail tablet
Manufactured by Roche
Sourced in Germany, Switzerland, United States, United Kingdom
PhosSTOP Phosphatase Inhibitor Cocktail Tablets are a ready-to-use solution designed to inhibit phosphatase activity in biological samples. The product contains a mixture of phosphatase inhibitors that effectively prevent the dephosphorylation of proteins, enabling the preservation of protein phosphorylation states for various analytical applications.
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Lab products found in correlation
Complete protease inhibitor cocktail tablet, by Roche (25 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
Complete mini protease inhibitor cocktail tablet, by Roche (7 mentions)
Ripa buffer, by Merck Group (6 mentions)
Complete protease inhibitor cocktail, by Roche (5 mentions)
Protease inhibitor cocktail tablet, by Roche (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Complete mini protease inhibitor, by Roche (4 mentions)
Odyssey fc imaging system, by LI COR (4 mentions)
Restore plus western blot stripping buffer, by Thermo Fisher Scientific (3 mentions)
Ecl plus, by Thermo Fisher Scientific (3 mentions)
β actin, by Merck Group (3 mentions)
Laemmli sample buffer, by Bio-Rad (3 mentions)
Immobilon pvdf membrane, by Merck Group (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ti tla 120.2 rotor, by Beckman Coulter (2 mentions)
N laurylsarcosine, by Merck Group (2 mentions)
T per, by Thermo Fisher Scientific (2 mentions)
87 protocols using phosstop phosphatase inhibitor cocktail tablet
1
Chromatin Immunoprecipitation (ChIP) of Hypothalamus Tissue
2
Protein Lysate Preparation from Zebrafish Tumors
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Protein lysates were prepared with mCherry expressed tumors from nf1a+/−; nf1b−/−; p53m/m fish. Briefly, mCherry-positive tumor mass were harvested from tricaine-anesthetized tumor fish, and were lysed on ice in 1 × RIPA (Cell Signaling, Danvers, MA, USA) containing, PhosSTOP phosphatase inhibitor cocktail tablet (Roche, Indianapolis, IN, USA) and complete protease inhibitor tablet (Roche). The inhibitors were prepared following the manufacturer's recommendation. Protein lysates were separated by gel electrophoresis, transferred to PVDF membranes and probed overnight at 4 °C with the following primary antibodies: anti-PDGFRA (Cell Signaling; 5241; 1:500), anti-p-tyrosine (EMD Millipore, Billerica, MA, USA; 05-321; 1:1000), anti-β-tubulin (Cell Signaling 2146; 1:2000), anti-AKT (Cell Signaling; 9272; 1:1000), anti-p-AKT (Cell Signaling; 4060; 1:1000), anti-p-ERK1/2 (Cell Signaling; 4377;1:1000) and anti-ERK1/2 (Cell Signaling; 9102; 1:1000). Primary antibody binding was visualized on X-ray film using anti-mouse-HRP (Cell Signaling; 7076; 1:10 000) or anti-rabbit-HRP (Cell Signaling; 7074; 1:10 000) secondary antibodies along with SuperSignal West Dura or Femto (Thermo Fisher Scientific, Waltham, MA, USA) chemiluminescent substrates. Stripping was performed using Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific) according to the manufacturer's protocol.
3
Protein Extraction and Western Blot Analysis
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For frozen tissue, lysates were prepared using a RIPA lysis buffer (50mM Tris (pH7.4), 150mM NaCl, 2mM EDTA, 1% Nonidet P-40, 50mM NaF, 0.1% SDS and 0.5% sodium deoxycholate with PhosStop Phosphatase-Inhibitor Cocktail tablet and protease-inhibitor cocktail tablet (Roche). The homogenate was centrifuged at 4°C for 10 min at 14,000g and the supernatant was used for western blot. The protein content of the supernatant was quantified using bicinchoninic acid reagents and BSA standards. Equal amounts of protein were separated using a polyacrylamide SDS-PAGE gel. After SDS-PAGE, proteins were transferred to PVDF membrane. The membrane was blocked for 1 h at room temperature followed by incubation overnight at 4°C with viperin antibody (Abcam), phospho-PKCμ, phospho-ACC, phospho-Akt, or GAPDH antibodies (Cell Signaling). After overnight incubation, the blots were incubated with HRP- conjugated secondary antibodies at a dilution of 1:5,000 for 1 h at room temperature. Bands were visualized by ECL plus (Thermo Scientific) according to the manufacturer's instructions and quantified using Image lab software.
4
Hippocampus Tissue Fractionation and Protein Extraction
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Hippocampus tissue (10–20 g) of mice or human brain tissue were homogenized in 10% (w/v) Tissue Protein Extraction Reagent (T-PER®, Thermo Fisher Scientific). The T-PER-insoluble pellet (P1) was sonicated with 10 volume of cold buffer H (10mM Tris-HCl, 1mM EGTA, 0.8 mM NaCl, 10% sucrose, pH 7.4) supplemented by 0.1mM PMSF (Sigma-Aldrich), PhosSTOP® Phosphatase Inhibitor Cocktail Tablet (Roche, 1 tablet per 10ml), Complete Protease Inhibitor Cocktail Tablet (Roche, 1 tablet per 10ml). After centrifugation at 28,000rpm in Beckman Ti TLA-120.2 rotor for 30min at 4°C, the supernatant (S1) was adjusted to 1% (w/v) N-laurylsarcosine (Sigma-Aldrich) and 1% (v/v) 2-mercaptoethanol (Sigma-Aldrich) and incubated at 37°C for 2 h with agitation (shaking). After centrifugation at 100,000rpm for 35 min at room temperature, the Sarkosyl-soluble supernatant (S2) was collected and resuspended in 1x lithium dodecyl sulfate (LDS) and sample reducing agent (RA) (Thermo Fisher Scientific). Sarkosyl-insoluble pellet (P2) was washed several times in 1% Sarkosyl in buffer H and the pellet was resuspended in 1x LDS/RA. Both samples (S2 and P2) were boiled at 95°C for 15 minutes for further western blot analysis.
5
Kidney Tissue Homogenization and Protein Analysis
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Homogenates were made from kidney cortex using cell extraction buffer (Thermo Fisher Scientific, FNN0011), containing a cOmplete protease inhibitor cocktail tablet (Roche, 4693159001) and PhosSTOP phosphatase inhibitor cocktail tablet (Roche, 4906837001). Protein concentrations were determined using Bradford Reagent (Bio-Rad, 5000001). Kidney homogenate (40 μg) was separated on 4%–12% gradient Tris–Glycine–SDS polyacrylamide gels, transferred to PVDF membranes, and proteins were detected by chemiluminescence substrate. Antibodies were purchased from Cell Signaling unless otherwise noted: inositol requiring enzyme-1α, #3294; extracellular receptor kinase (ERK), #4695; phosphorylated-ERK(p-ERK), #4370; cleaved caspase 3 (CC3), #9664; C/EBP homologous protein, #2895; c-jun n-terminal kinase (JNK), #9258; phosphorylated-JNK (p-JNK), #4668; proliferating cell nuclear antigen (PCNA), #13110; phosphorylated-eukaryotic initiation factor 2α, #3398; sequestosome 1/p62 (p62), #5114; microtubule-associated protein light chain 3, #3868; α-tubulin (Santa Cruz Biotechnology, sc-5286) and β-actin (Sigma-Aldrich, A2228).
6
Quantification of Hepatic INHBE Levels
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Liver tissue samples were homogenized using a Polytron homogenizer in RIPA buffer (Merck Millipore, Billerica, MA, USA) containing a Complete® Mini EDTA-free cocktail tablet (Roche Diagnostics, Basel, Switzerland) and PhosSTOP phosphatase inhibitor cocktail tablet (Roche Diagnostics). After sonication with a Bioruptor (Cosmo Bio, Tokyo, Japan), the tissue lysates were further solubilized by continuous stirring for 1 h at 4°C and centrifuged to remove insoluble material. The supernatants (6.6 μg of protein per sample) were used for measurement of hepatic INHBE protein levels. The concentrations of INHBE in the liver and plasma were determined using an ELISA kit for mouse INHBE (Wuhan USCN Business, Houston, TX, USA) according to the manufacturer’s instructions.
7
Western Blot Analysis of p53 in Hepa1-6 Cells
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In total 1 × 106 Hepa1-6 cells were plated per well of a 6-well plate in DMEM complete media and treated with complete media only, 20 µM Nutlin-3 or 10 µM Erastin for 24 h. Cells were lysed in SDS lysis buffer (2% SDS, 50 mM Tris-HCl, 5% glycerol, 5 mM EDTA, 1 mM NaF, 10 mM β-glycerophosphate, 1 mM PMSF, 1 mM activated Na3VO4, 1 mM DTT, 1% phosphatase inhibitor cocktail 2 (Sigma-Aldrich), 1% PhosSTOP Phosphatase Inhibitor Cocktail Tablet (Roche)), filtered overnight at 3000 rpm through AcroPrep Advance Filter Plates (Pall Corporation), and stored at −80 °C. Western blots were performed according to manufacturer instruction with the iBlot Dry Transfer System (Life Technologies, Carlsbad, CA, USA) using an antibody to p53 (Clone 1C12; Cell Signaling Technology) and then normalized to signal from an anti-β-actin (Cell Signaling Technology). Signals from immunoblots were detected using either an Alexa 680-conjugated anti-rabbit antibody or an Alexa 800-conjugated anti-mouse antibody (LI-COR Biosciences). Membranes were visualized using an Odyssey infrared imaging system (LI-COR Biosciences).
8
Stable Expression of Tagged β2AR in HEK293
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HEK293 clones stably expressing human β2AR with N-terminal Flag-tag, C-terminal eGFP-tag (Flag-hβ2AR-eGFP) were created via transfection of HEK293 cell with plasmid harboring the coding sequence for Flag-hβ2AR-eGFP. Cells with membrane eGFP were selected (under fluorescence microscope) and then subcultured for these experiments. HEK293 clones expressing the tagged β2AR were grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% (v/v) fetal bovine serum, 100 μg/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine, and maintained in 5% CO2/95% air at 37°C. Cells were grown to 90% confluence in 100 mm-dishes, then washed and replenished with DMEM pre-warmed to 37°C. Beta-adrenergic agonists or antagonists were added cell culture media to a final concentration of 10 μM (unless otherwise specified). The cells were incubated for indicated times. Control dishes were incubated with DMEM only, without agonists or antagonists. Cells were harvested with 120 μl cell lysis buffer (phosphate-buffered saline containing 10 mM NaF, 10 mM sodium pyrophosphate, 0.2 mM sodium vanadate, 1% n-dodecyl-β-D-maltoside, 2 × protease inhibitor tablet (EDTA-free, Roche, Nutley, NJ), 2 × phos-STOP phosphatase inhibitor cocktail tablet (Roche, Nutley, NJ). Whole cell lysates were stored at −80°C prior to analysis.
9
Immunoprecipitation of HA-tagged Proteins
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Pierce™ Anti-HA Magnetic Beads were washed with 0.05% (v/v) TBST and then with lysis buffer prior to addition of 4 mg cleared cell lysate (at a ratio of beads to protein of 1:2000). Beads and lysate were incubated overnight using an end-over-end rotor at 4°C. Beads were then collected using a magnetic stand and washed three times with wash buffer (40 mM Tris-HCl pH 8.0, 0.1% (v/v) NP-40, 1 mM EGTA, 6 mM EDTA, 6 mM DTT, 0.5 M NaCl, 1× PhosSTOP™ phosphatase inhibitor cocktail tablet (Roche), 1× cOmplete™ Protease Inhibitor Cocktail (Roche)); one time with HPLC grade water and finally, three times with 25 mM ammonium bicarbonate (AMBIC). To recover the immunoprecipitated material, the beads were resuspended in 100 µl of 25 mM AMBIC to which 6 µl of a 1% (w/v) solution of RapiGest (Waters, UK) in 25 mM AMBIC was added. Samples were then heated to 80°C for 10 min. Supernatants containing the eluted proteins were recovered using a magnetic stand and used for in-solution digestion.
10
Protein Extraction from Tissue Samples
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