Cells were washed with ice-cold phosphate-buffered saline (PBS) buffer (Invitrogen; 20012050) and lysed with harvest buffer (10 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [HEPES; pH 7.9], 50 mM sodium chloride [NaCl], 500 mM sucrose, 0.1 mM ethylenediaminetetraacetic acid [EDTA], and 0.5% Triton X-100) supplemented with 1% Halt inhibitors (Thermo Fisher Scientific; 78443) for 10 min. The cell lysate was centrifuged at 3000 rpm for 3 min at 4 °C, and the supernatant containing cytosolic proteins was transferred to a new tube. The pellet containing nuclear proteins was dissolved with nuclear lysis buffer (10 mM HEPES [pH 7.9], 0.5 M NaCl, 0.1 mM EDTA, 0.1 mM egtazic acid, and 0.1% NP40) supplemented with 1% Halt inhibitors (Thermo Fisher Scientific; 78443) for 5 min at 4°C. Then, the solution was sonicated at 4°C for six cycles (1 cycle = 30 s sonication [high intensity] and 30 s cooldown) using a Bioruptor Plus sonication (Diagenode; UCD-300). After sonication, the solution was centrifuged at 15,000 rpm for 10 min, and the supernatant was retained for nuclear extraction. Protein concentration was determined with the BCA assay (Thermo Fisher Scientific, 23227). Immunoblotting was performed as previously described (4 (link)). All the blots are representative of at least two independent experiments.
Halt inhibitors
Manufactured by Thermo Fisher Scientific
Sourced in United States
HALT inhibitors are laboratory equipment designed to mitigate the effects of Highly Accelerated Life Testing (HALT) on sensitive electronic components or systems. Their core function is to protect against the damaging effects of environmental stresses, such as temperature, humidity, and vibration, during the HALT process.
Automatically generated - may contain errors
Lab products found in correlation
Bca assay, by Thermo Fisher Scientific (1 mentions)
Bioruptor plus sonication, by Diagenode (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Tissuerupter, by Qiagen (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Odyssey ir scanner, by LI COR (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Fer 1, by MedChemExpress (1 mentions)
5 protocols using halt inhibitors
1
Nuclear Protein Extraction Protocol
2
Skin Biopsy Protein and DNA Extraction
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Skin biopsies (3 mm) were incubated in 50 ml conical tubes with 5 ml of DMEM with or without serum and 125 nM ARQ 092 for 96 hours at 37 °C. Tissues were frozen on dry ice and stored at −80 °C for 2 days. Each biopsy was dissected such that protein was extracted from approximately 2/3 and DNA was extracted from the remainder. Protein was isolated by homogenizing the tissue in 500 μl 1× cell lysis buffer (Cell Signaling Technologies, Danvers, MA, USA) supplemented with HALT inhibitors (Thermo Scientific, Waltham, MA, USA) using a TissueRupter (Qiagen, Valencia, CA, USA) for 2 minutes on ice. Lysates were vortexed for 10 minutes followed by centrifugation for 10 minutes at 21,000 rcf at 4 °C. The cleared lysate was transferred to a new tube and frozen at −80 °C. DNA was extracted by standard methods and mutation levels were measured by a custom restriction fragment length polymorphism assay as described3 (link).
3
Brain Tissue Processing for Neurochemical Analyses
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Anesthetized animals were sacrificed by cervical dislocation. Brains were immediately removed and kept on ice for dissection of PFC and HIPP, according to the atlas of Paxinos and Franklin. Tissues were frozen and stored at −80 °C until analyses were carried out. Samples (PFC and HIPP) were homogenized (1:10 w/v) at 4 °C using a PBS buffer containing a 1:100 protease and phosphatase inhibitor cocktail (HALT inhibitors, Thermo Fisher Scientific, Cleveland, OH, USA) for biochemical analyses or perchloric acid 0.1 M for neurochemical analyses. Homogenates were centrifuged at 13,000× g at 4 °C for 20 min, and supernatants were used for further determinations.
4
Western Blot Protocol for Protein Analysis
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Cells (1 × 106) were grown to 70–80% confluency and treated with DMSO control, (1S,3R)-RSL3 (RSL3, Catalog # 19,288, Cayman Chemical, MI) or Fer1 (Medchemexpress, NJ) as per the experiments. Cells were washed with cold PBS and total proteins were extracted with RIPA buffer containing Halt inhibitors (Thermofisher, MA). A 20–30 μg of total protein were resolved on 4–12% NuPAGE Bis–Tris gels (Thermofisher) and transferred to nitrocellulose membrane using a semi-dry iBlot2 kit (Thermofisher). Blots were blocked with 5% milk in PBS-T (0.01% Tween-20 in phosphate buffered saline). Blots were incubated overnight at 4 °C with primary antibodies and 2 h with HRP-conjugated secondary antibodies at room temperature. Blots were developed using ECL kit (Thermofisher) or scanned using the OdysseyIR scanner (Li-CORBiosciences, NE). All antibodies utilized for Western blotting are listed in Supplementary Table 1 . Western blot density quantification was performed using ImageJ software36 (link) and are displayed in Supplementary Fig. 1 . Some of these Western blots presented in the study were processed using either the mouse or rabbit IRdye conjugated secondary antibodies and scanned with OdysseyIR scanner (Li-CORBiosciences, NE).
5
Brain Region Dissection and Preparation
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After behavioural tests, the mice were sacrificed by cervical dislocation and brains were randomly divided for ex vivo analysis. PFC, HIPP, HYP and AMY were removed from brains, in accordance with the mouse brain atlas of Paxinos and Franklin, and frozen, stored at −80 °C for subsequent analyses. To perform biomolecular studies, the tissues were homogenated and diluted 1:10 w/v in PBS buffer with 1:100 protease and phosphatase inhibitor (HALT inhibitors, Thermo Fisher Scientific, Cleveland, OH, USA) at 4 °C; while for neurochemical analyses, the samples were homogenated and diluted 1:10 w/v in perchloric acid 0.1 M at 4 °C. In both cases, after dilution, a centrifuge at 10.000 x g at 4 °C for 10 min was carried out and the supernatants were analysed.
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