Nuclear and cytoplasm extract were obtained from HPC treated with DMSO or 100 nM of BMS-595 for 24 h. Nuclear protein was extracted using CelLytic NuCLEAR Extraction Kit (Sigma) with phosphatase inhibitor cocktail (Sigma). To retrieve the cytoplasm lysate, the cells were lysed with RIPA buffer (Sigma) supplemented with a protease inhibitor cocktail (Sigma) and a phosphatase inhibitor cocktail. Denatured protein samples were separated by electrophoresis with 25 μg/lane and transferred onto PVDF membrane. The list of antibodies provided in Supplemental Table 1 . The detected proteins were visualized by the ECL system (GE Healthcare Life Sciences).
Phosphatase inhibitor cocktail
Manufactured by Merck Group
Sourced in United States, Germany, Switzerland, China, Japan, Italy, Canada, Macao, Denmark, Hungary, India, Sao Tome and Principe, Spain, Ireland, United Kingdom
The Phosphatase inhibitor cocktail is a laboratory product designed to inhibit the activity of phosphatase enzymes. Phosphatases play a crucial role in various cellular processes, and their inhibition can be valuable in biological research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (423 mentions)
Protease inhibitor cocktail, by Roche (114 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (62 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (48 mentions)
Ripa buffer, by Merck Group (41 mentions)
Pvdf membrane, by Merck Group (39 mentions)
Pvdf membrane, by Bio-Rad (38 mentions)
Protease inhibitor, by Merck Group (35 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (33 mentions)
β actin, by Merck Group (31 mentions)
Protease inhibitor, by Roche (31 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (25 mentions)
Nitrocellulose membrane, by Bio-Rad (25 mentions)
β actin, by Cell Signaling Technology (24 mentions)
Bradford assay, by Bio-Rad (21 mentions)
Proteinase inhibitor cocktail, by Merck Group (20 mentions)
Ripa buffer, by Thermo Fisher Scientific (20 mentions)
Complete protease inhibitor cocktail, by Roche (19 mentions)
Gapdh, by Cell Signaling Technology (19 mentions)
Polyvinylidene difluoride membrane, by Merck Group (19 mentions)
1 110 protocols using phosphatase inhibitor cocktail
1
Protein Extraction and Western Blot
2
Brain Tissue Fractionation and Astrocyte Lysis
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Mice were anesthetized with Avertin (tribromoethanol, 250 mg/kg) and perfused transcardially with 0.9% saline. Brains were removed, microdissected in ice-cold PBS, and homogenized in ice- cold buffer containing 1X PBS (pH 7.4), 1 mM 1,4-Dithiothreitol (DTT, Roche), 0.5 mM EDTA, 0.5% Triton X-100, 0.1 M phenylmethyl sulfonyl fluoride (PMSF), a protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (Sigma-Aldrich). Homogenates were sonicated twice for 5 min at 40 Amp and centrifuged at 10,000 rpm for 10 min at 4°C. The supernatants were collected for measurement of protein concentrations and western blotting.
Astrocyte cultures were lysed on ice for 10 min using a lysis buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 0.5% deoxycholate, 5 mM EDTA, 0.2% Triton X-100, a protease inhibitor cocktail (Roche) and a phosphatase inhibitor cocktail (Sigma-Aldrich). The lysates were sonicated for 1 min at 40 Amp and centrifuged at 10,000 rpm for 10 min at 4°C. The supernatants were collected for measurement of protein concentrations and western blotting.
Astrocyte cultures were lysed on ice for 10 min using a lysis buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 0.5% deoxycholate, 5 mM EDTA, 0.2% Triton X-100, a protease inhibitor cocktail (Roche) and a phosphatase inhibitor cocktail (Sigma-Aldrich). The lysates were sonicated for 1 min at 40 Amp and centrifuged at 10,000 rpm for 10 min at 4°C. The supernatants were collected for measurement of protein concentrations and western blotting.
3
HA-Immunoprecipitation of Transfected MCM Proteins
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HEK293T were transiently transfected with pMSCV-FLAG-HA-MCM8, -MCM8 L387E, -MCM9, -MCM9 M45E or -GFP control (Transporter 5, Polysciences 26008-5) and harvested 3 days later for HA-immunoprecipitation33 (link). Briefly, cell pellets were resuspended in mammalian cell lysis buffer (MCLB) (50 mM Tris-HCl pH 7.5, 1% IGEPAL CA-630) supplemented with 150 mM NaCl, a protease inhibitor (Goldbio, GB-331) and phosphatase inhibitor cocktail (Sigma, 4906837001). Following incubation for 30 minutes at 4 °C, extracts were cleared by centrifugation and supernatants collected. The remaining pellets were subjected to a second round of extraction by resuspending them in MCLB supplemented with 500 mM NaCl and protease (Goldbio, GB-331) and phosphatase inhibitor cocktail (Sigma, 4906837001), and incubating them for an additional hour at 4 °C. After clearing these extracts by centrifugation, the NaCl concentration of the collected supernatants was adjusted to 150 mM with MCLB and combined with the supernatants from the first round of extraction. Combined lysates were then incubated with anti-HA agarose beads (Sigma, A2095) for 4 hours at 4 °C. After incubation, beads were washed four times in MCLB buffer supplemented with 150 mM NaCl, and then boiled in LDS (lithium dodecyl sulfate) sample buffer (Thermo Fisher, NP0008) supplemented with β-mercaptoethanol to elute the bound proteins.
4
PTEN C-terminus Interaction Analysis
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Association of PTEN and its C-terminal tail (PTEN352-403-YFP-FLAG) was analyzed, as previously described21 (link)30 . HEK293 cells were transiently transfected with a plasmid carrying PTEN352-403-YFP-FLAG and then cultured overnight. Cells were lysed in lysis buffer containing 1% Nonidet P-40, 50 mM NaCl, 20 mM Tris-HCl (pH 7.5), 10% glycerol, 0.1 mM EDTA, phosphatase inhibitor cocktail (Sigma) and protease inhibitor cocktail (Roche). The lysates were then cleared by centrifugation at 13,000 rpm for 20 min at 4 °C. Dictyostelium cells expressing GFP fused to wild-type and mutant PTEN were lysed in 1% Nonidet P-40, 300 mM NaCl, 10 mM Tris-HCl (pH 7.5), 2 mM EDTA, phosphatase inhibitor cocktail (Sigma) and protease inhibitor cocktail (Roche). The lysates were then cleared by centrifugation at 13,000 rpm for 20 min at 4 °C. 100 μl of the HEK293 cell lysates were mixed with 500 μl of the Dictyostelium cell lysates. 15 μl of beads coupled to anti-FLAG antibodies (Sigma) were added to the mixtures and incubated for two hours. The bound fractions were analyzed by SDS-PAGE and immunoblotting using antibodies to GFP, which recognize both GFP and YFP.
5
Brain Tissue Fractionation and Astrocyte Lysis
6
Protein Sample Preparation and Lysis
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Protein samples were prepared from equal numbers of cells grown in 10-cm culture dishes (Corning Inc., Corning, NY). Cells were washed three times with ice-cold PBS, scraped into ice-cold PBS (1 ml/culture dish), transferred into 2-ml tubes, and centrifuged at 200 g for 5 min at 4°C. The cell pellets were suspended in ice-cold lysis buffer containing either DDM [50 mM sodium phosphate (pH 7.4), 1% DDM, 1 mM DTT, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma)] or Triton X-100 [50 mM Tris/HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM DTT, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma)], vortexed vigorously for 10 s, and incubated on ice for 30 min. The tubes were then centrifuged at 20000 g for 10 min at 4°C. The supernatants were transferred into new precooled 1.5-ml microtubes and total protein concentration of the lysates was determined by the Pierce 660-nm protein assay (Pierce, Rockford, IL). The samples were aliquoted and stored at −80°C until further analysis.
7
Mammalian Cell Transfection and Lysis
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293T cells or C2C12 cells were seeded in 100 mm dishes overnight prior to transfection using 1:3 ratio of DNA to JetPrime reagent (Polyplus) in JetPrime Buffer (Polyplus) at approximately 80% confluency, according to the manufacturer’s protocol. Cells were scraped and lysed 24 hr following transfection in native cell lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM beta-glycerophosphate, 1% Triton X-100) containing freshly added protease and phosphatase inhibitors (1 mM PMSF, 1× protease inhibitor cocktail, 1× phosphatase inhibitor cocktail; Sigma) for 20 min on ice. For denaturing lysis to prepare samples for acetyl-lysine pulldown, homogenized tissue samples or cells were lysed in RIPA lysis buffer (25 mM Tris pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing freshly added protease and phosphatase inhibitors (1 mM PMSF, 1× protease inhibitor cocktail, 1× phosphatase inhibitor cocktail; Sigma). Whole-cell lysates were cleared by centrifugation at 15,000 rpm for 20 min. Protein quantification was performed using BCA kit (Pierce), following the manufacturer’s instructions.
8
Protein Extraction from Hippocampal Tissue
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The protein was extracted as previously described with some modifications [25 (link)]. In short, 40 mg of frozen hippocampal tissue was excised on ice before adding 500 μL RIPA buffer (50 mmol/L tris pH 8.0, 150 mmol/L sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate) with 1:100 protease inhibitor cocktail (Sigma-Aldrich, Darmstadt, Germany) and 1:100 phosphatase inhibitor cocktail (Sigma-Aldrich, Darmstadt, Germany) and homogenized by mortar and pestle on ice. The samples were centrifuged for 10 min at 12,000 rpm at 4 °C and the supernatant divided in aliquots and stored at −80 °C. In addition, another 10 mg of hippocampal tissue was excised on ice before adding 250 μL of Tissue Protein Extraction Reagent (T-PER) (Thermo Fisher Scientific, Waltham, MA, USA) with 1:100 protease inhibitor cocktail (Sigma-Aldrich, Darmstadt, Germany) and 1:100 phosphatase inhibitor cocktail (Sigma-Aldrich, Darmstadt, Germany). The samples were centrifuged at 10,000× g for five minutes at 4 °C according to manufacturer’s instructions and the supernatant divided in aliquots and stored at −80 °C. Two animals from the CTRL group were excluded for technical reasons, leaving the CTRL group size at n = 13. Protein concentrations were determined using a commercial BCA kit according to manufacturer’s instructions (Merck Millipore, Darmstadt, Germany).
9
Protein Extraction and Western Blot Analysis
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Protein lysates were prepared from ARVMs by homogenisation in lysis buffer (in mM: EGTA 1, DTT 1, Tris-HCl 10, NaCl 140; pH 7.6 adjusted with NaOH and supplemented with 1x phosphatase inhibitor cocktail and 1x protease inhibitor cocktail (both from SIGMA)) and subsequent incubation on ice for 30 min. For immunoblotting of cMyBP-C, cells were lysed in a modified RIPA buffer [in mM: NaCl 150, Tris-HCl 20, EDTA 10, DTT 1 and 1% each of sodium deoxycholate and TRITON X-100 and 0.1 % SDS; pH 7.6 adjusted with NaOH and supplemented with 1x phosphatase inhibitor cocktail and 1x protease inhibitor cocktail (both from SIGMA)]. Cell lysates were collected after centrifugation at 25,000 g for 15 min at 4 °C. Equivalent amounts of protein, as determined by BCA protein assay (Pierce), were separated on a 4-12% BIS-TRIS, SDS-PAGE (NuPage, NOVEX), and analysed by standard immunoblotting techniques [Enhanced Chemiluminescence using HRP conjugated secondary antibodies (Figures 3,5 and Supplementary Figures 6 and12; II) and fluorescence detection using fluorescently labelled secondary antibodies and imaged on a Licor Odyssey CLx imaging system (Figure 6 and Supplementary Figure 12; III) as previously described [60] (link). The following primary antibodies were used:
10
Protein Extraction and Western Blot Analysis
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