For cytoplasmic and nuclear fractionation, AML12 cells were grown to 100% confluency, washed twice with ice-cold PBS and incubated with buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, pH 7.9, PMSF 50 µg ml−1, Na-Orthovanadate 1 mM, DTT 1 mM, NP-40 (0.6%), HALT-Phosphatase inhibitor (Thermoscientific) 1:1000, complete Protease-inhibitor tablet (Thermoscientific)) on ice for 15 min. Cells were collected and centrifuged for 5 min at 14,000 × g. Supernatant (cytoplasmic fraction) was collected and saved. The pellet was processed further for nuclear extraction by washing with buffer A without NP-40, followed by resuspension in buffer C (10 mM HEPES, 0.4 mM NaCl, 1 mM EDTA, 1 mM EGTA, pH 7.9, PMSF 50 µg ml−1, Na-Orthovanadate 1 mM, DTT 1 mM, NP-40 (0.6%), HALT-Phosphatase inhibitor (Thermoscientific) 1:1000, complete Protease-inhibitor tablet (Thermoscientific)) and incubation on ice for 15 min, vortexing every minute. Fraction was centrifuged at 14,000 × g for 8 min and supernatant (nuclear fraction) collected.
Halt phosphatase inhibitor
Manufactured by Thermo Fisher Scientific
Sourced in United States, Switzerland
Halt phosphatase inhibitor is a laboratory reagent designed to inhibit the activity of phosphatases, enzymes that remove phosphate groups from molecules. It is commonly used in biochemical and cell biology research to preserve the phosphorylation state of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Halt protease inhibitor, by Thermo Fisher Scientific (21 mentions)
Ripa buffer, by Thermo Fisher Scientific (12 mentions)
Bca assay, by Thermo Fisher Scientific (10 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (8 mentions)
Ripa buffer, by Merck Group (8 mentions)
Complete protease inhibitor, by Roche (7 mentions)
Protease inhibitor, by Merck Group (7 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor, by Roche (6 mentions)
Odyssey blocking buffer, by LI COR (4 mentions)
Pierce lane reducing sample buffer, by Thermo Fisher Scientific (4 mentions)
β actin, by Merck Group (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Bradford assay, by Bio-Rad (3 mentions)
Pierce universal nuclease, by Thermo Fisher Scientific (3 mentions)
Cell lysis buffer, by Cell Signaling Technology (3 mentions)
Bis tris gel, by Thermo Fisher Scientific (3 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
97 protocols using halt phosphatase inhibitor
1
Cytoplasmic and Nuclear Fractionation
2
Protein Extraction and Western Blot Analysis
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Cells were lysed in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and Halt™ phosphatase inhibitor (Thermo Fisher Scientific). The lysates were incubated on ice for 20 min and collected by centrifugation at 13,000 rpm for 20 min at 4°C. The supernatants were removed to clean tubes, mixed with 5× sample buffer, and heated at 100°C for 5 min. Proteins were separated by SDS-PAGE through 10% gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were blocked in phosphate-buffered saline (PBS) containing 5% (w/v) bovine serum albumin (BSA, Bioworld, Dublin, OH, USA) and 0.05% (v/v) Tween 20 (PBST) for 1 h and incubated overnight at 4°C with primary antibody against STAT3 or phospho-STAT3 (Cell Signaling Technology, Danvers, MA, USA). Antibody against GAPDH (sc-25778, Santa Cruz Biotechnology) was used as internal control. Membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies (SC-2005, Santa Cruz Biotechnology) for 1 h and visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore).
3
Glioblastoma Tissue Protein Extraction
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GBM primary tumor samples were homogenized and powdered in liquid nitrogen using a mixer mill (Retsch Inc., Haan, Germany). Samples were maintained at liquid N2 temperature throughout the process. Homogenized and powdered tissue samples were then re-suspended in 5X volume of the weight of the tissue sample in a reducing buffer (125 mM Tris pH 6.8, 4% SDS (w/v), 10% glycerol (v/v), 5% 2-mercaptoethanol (v/v), complete protease inhibitor (Roche Diagnostics Corp., Indianapolis, IN, USA), HALT phosphatase inhibitor (Thermo Fisher Scientific, Grand Island, NY, USA). Samples were then heated to 70°C with mixing at 1,400 rpm for 10 min, sonicated with a tip sonicator for 30 sec at power level 4, and then centrifuged at 16,000 × g for 10 min at room temperature. The supernatant was then collected.
4
Protein Extraction and Western Blot Analysis
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Flash-frozen tumors were homogenized in PBS with Halt phosphatase inhibitor (Thermo Fisher Scientific) and protease inhibitor (Sigma-Aldrich) using the Precellys Evolution Homogenizer with Cryolys (Bertin Instruments). The preset elastic setting was used for homogenizing. Tumor homogenates were centrifuged to remove PBS and resuspended in RIPA buffer with phosphatase and protease inhibitors. Lysates were sonicated 2 × 10 s using a Branson digital sonifier at 10% amplitude. Samples were centrifuged at 15,000 rpm for 5 min at 4°C and supernatants collected and quantified by Bradford assay (Bio-Rad). Samples (35 μg total) were blotted for BRAF (sc-5284; 1:500; Santa Cruz) and β-Actin (#3700; 1:1,000; Cell Signaling) and imaged using a LI-COR Odyssey CLx system. Bands were quantified using Image Studio Version 5.2 software (LI-COR Biosciences).
5
Detergent-Based Brain Protein Extraction
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Brain homogenates [10% (w/v)] were prepared in PBS using a Minilys homogenizer and CK14 homogenizing tubes. Nine volumes of brain homogenate were then mixed with one volume of 10X detergent buffer [5% (v/v) Nonidet P-40, 5% (w/v) sodium deoxycholate in PBS] containing Pierce Universal Nuclease (ThermoFisher #88701) and Halt Phosphatase Inhibitor (ThermoFisher #78420), and then incubated on ice for 20 min. Samples were clarified by centrifugation at 1000x g for 5 min at 4 °C to generate detergent-extracted brain homogenate. For thermolysin (SigmaAldrich #T7902) digestions, extracts were treated with 50 μg/mL protease (TL:protein ratio of 1:100) at 37 °C for 1 h with constant agitation (600 rpm). Digestions were terminated by the addition of ethylenediaminetetraacetic acid (EDTA) to a final concentration of 5 mM. For PK digestions, detergent-extracted brain homogenates were treated with 100 μg/mL PK (PK:protein ratio of 1:50) at 37 °C for 1 h with constant agitation (600 rpm). Digestions were terminated by the addition of PMSF to a final concentration of 4 mM. Following digestion, samples were ultracentrifuged at 100,000x g for 1 h at 4 °C. The supernatant was discarded, and the pellets were resuspended by boiling in loading buffer. Samples were then analyzed by SDS-PAGE followed by immunoblotting.
6
Western Blot Analysis of Cerebellar and B-Cell Proteins
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Mouse cerebellar tissues and B-lymphocyte cell pellets were homogenized with a cell lysis buffer (Cell Signaling) with Halt phosphatase inhibitor (Thermo-Fisher), complete protease inhibitors (Roche) and PSMF (Sigma-Aldrich). Thirty micrograms of lysates were loaded into 4–12% Bis–Tris gels (Invitrogen). Electrophoresis was carried out according to the manufacturer's recommendations. Following electrophoresis, the proteins were transferred to nitrocellulose membranes by the iBlot device (Invitrogen), blocked with an Odyssey blocking buffer (LI-COR Biotechnology) for 1 h. Membranes were incubated overnight with the following primary antibodies in blocking buffer: COX1 (ab133319 for human, ab133319 for mouse; Abcam), COX2 (ab62331 for human, ab6665 for mouse; Abcam), phospho-CREB (4095), phospho-cJun/AP1 (5464), phospho-NFκB (4025; Cell signaling), cPLA2 (sc-454), phospho-cPLA2 (sc-34391; Santa Cruz Biotech, Santa Cruz, CA, USA), β-actin (A5441; Sigma-Aldrich), and β-tubulin (DSHB-E7; DSHB, Iowa). Subsequently, the membranes were incubated with a corresponding pair of IRDye 680CW and IRDye 800CW-coupled secondary antibodies (LI-COR). Proteins were visualized with the Odyssey infrared imager and software (LI-COR) according the manufacturer's instruction.
7
Protein Extraction and Western Blotting
8
Western Blotting for CLIP Protein Expression
9
Western Blot Analysis of Stem Cells
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Samples were analyzed by western blotting as described previously.25 (link),55 (link) Cells were washed with ice-cold PBS, and total protein extract was prepared with RIPA buffer with 1× Halt protease inhibitor cocktail and Halt phosphatase inhibitor (Thermo Fisher Scientific) at a ratio of 5 × 106 cells/mL. 30 μg protein for iPSC-derived OCs or 15 μg for iPSC-LESCs was loaded onto 4%–15% Mini-PROTEAN TGX gels (Bio-Rad, CA, USA) and transferred to an Immun-Blot polyvinylidene fluoride (PVDF) membrane using a Trans-Blot SD semi-dry transfer cell (Bio-Rad). Membranes were blocked with 5% non-fat dry milk in PBS-Tween 20 (0.1%) for 2 h and incubated overnight at 4°C with the following primary antibodies diluted in blocking buffer: PAX6 (1:2,000, Covance), ABCG2 (1:1,000, Santa Cruz Biotechnology), and β-actin (1:5,000, Sigma-Aldrich). Incubation with horseradish peroxidase-conjugated secondary antibody (anti-mouse or -rabbit, 1:5,000, Applied Biosystems) was done for 2 h at room temperature. Membranes were incubated with Clarity Western ECL Substrate (Bio-Rad) and imaged using the ChemiDoc XRS Imaging System (Bio-Rad). Band intensities were quantified using the Fiji/ImageJ software (National Institutes of Health, MD, USA).
10
Western Blot Analysis of Akt and pAkt
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Cells were lysed with RIPA buffer (Sigma–Aldrich, Taufkirchen, Germany) containing Complete™ protease inhibitor cocktail (Thermo Fisher Scientific, Dreieich, Germany), HALT™ protease inhibitor and HALT™ phosphatase inhibitor (Thermo Fisher Scientific, Dreieich, Germany). Western blot was performed with proteins from whole cell lysates. Primary antibodies: anti-Akt and anti-pAkt (Ser473) (Cell Signaling, Frankfurt a. M., Germany); anti-β-actin (Abcam, Cambridge, UK). Secondary antibodies: goat anti-rabbit and goat anti-mouse (Santa Cruz, Heidelberg, Germany). For detection, Western blotting Luminol Reagent (Santa Cruz, Heidelberg, Germany), and Amersham Hyperfilm™ ECL (GE Healthcare Limited, Buckinghamshire, UK) were used. Densitometry was done using ImageJ 1.48v (Wayne Rasband (National Institutes of Health), MD, USA).
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