The reagents that were used in the experiments are listed below. (1) Sigma-Aldrich, Saint Louis, MO, USA: Poly(ethyleneimine) solution (Cat. no. P3143), penicillin–streptomycin (Cat. no. P4333), L-Glutamine (Cat. No G85402). (2) Life Technologies, Grand Island, NY, USA: B-27 supplement (Cat. no. 17504044), Trypsin 2.5% (Cat. no. 15090046). (3) Molecular Probes, Eugene, OR, USA: Fura-2 AM (Cat. no. F1221). (4) Tocris Bioscience, Bristol, UK: UBP 310 (Cat. no. 3621), 5-Nonyloxytryptamine oxalate (Cat. No. 0901), WIN 55,212-2 mesylate (Cat. no. 1038) (5) Alomone Labs, Jerusalem, Israel: D-AP5 (Cat. no. D-145), NBQX (Cat. no. N-185). (6) Cayman Chemical, Ann Arbor, MI, USA: Bicuculline (Cat. no. 11727), HEMADO (Cat. No. 21015); (7) AppliChem, Darmstadt, Germany: EDTA (Cat. no. A5097), EGTA (Cat. no. A-0878). (8) Dia-M, Moscow, Russian Federation: HEPES (Cat. no. 3350). (9) Abcam, Cambridge, UK: N6-Cyclohexyladenosine (Cat. no. ab120472). (10) Paneco, Moscow, Russian Federation: Neurobasal-A medium.
Ethylene glycol tetraacetic acid (egta)
Manufactured by AppliChem
Sourced in Germany
EGTA is a chemical compound that functions as a chelating agent. It is commonly used in biochemical and cell biology applications to bind and sequester calcium ions (Ca2+) from aqueous solutions.
Automatically generated - may contain errors
Lab products found in correlation
Polyethyleneimine solution, by Merck Group (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Trypsin 2.5, by Thermo Fisher Scientific (2 mentions)
Bicuculline, by Cayman Chemical (2 mentions)
Hepes, by Dia-M (2 mentions)
Neurobasal a medium, by PanEco (2 mentions)
Fura 2 am, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Ubp 310, by Bio-Techne (1 mentions)
Ethylene diamine tetraacetic acid (edta), by AppliChem (1 mentions)
D ap5, by Alomone (1 mentions)
Stainless steel bead, by Qiagen (1 mentions)
Igepal, by Merck Group (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Dithiothreitol (dtt), by AppliChem (1 mentions)
Sodium chloride (nacl), by Avantor (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Sodium deoxycholate, by Carl Roth (1 mentions)
5 protocols using ethylene glycol tetraacetic acid (egta)
1
Neuronal Cell Culture Reagents
2
Quantifying Tissue Protein Extracts
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Total proteins were extracted from tissue samples using 10 µL tris‐lysis buffer (150 mM NaCl (cat. no. 1.06404.1000, VWR), 20 mM Tris (cat. no. A1087, AppliChem GmbH, Darmstadt, Germany), 1mM EGTA (cat. no. E3889, Sigma‐Aldrich), 1% (v/v) IGEPAL (v/v; cat. no. 18896, Sigma‐Aldrich) and 1 mM EDTA (cat. no. 03690, Sigma‐Aldrich)) with 2% Halt protease inhibitor cocktail (v/v) (cat. no. 78438, Thermo Fisher Scientific) per mg tissue. Samples were homogenised using stainless steel beads (Qiagen, Hilden, Germany) and the TissueLyser II (Qiagen) at 30 cycles/s for 2 min. Samples were incubated on ice for 20 min and mixed by vortexing every 5 min. Samples were centrifuged at 15,000g for 20 min at 4°C, and the supernatants were stored at −80°C until analysis. The whey protein BLG were quantified in tissue extracts using a commercial bovine BLG ELISA kit (cat. no. A10‐125A, Bethyl Laboratories, Montgomery, AL, US) according to the manufacturer's protocol with the exception that plates were coated overnight.
3
Nuclei Extraction from Cultured Cells
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To separate nuclei from cytoplasm in CNs, an Abcam protocol was applied with slight modifications. In brief, cells were scraped off on ice in fractionation buffer [20 mM HEPES (pH 7.4) (Carl Roth), 10 mM KCl (Carl Roth), 2 mM MgCl2 (Merck Millipore), 1 mM EDTA (Carl Roth), 1 mM EGTA (AppliChem), 1 mM dithiothreitol (AppliChem) and 1×PI] and incubated for 15 min on ice. Samples were then passed through a 27-gauge needle (Sigma-Aldrich) ten times, then incubated on ice for 20 min. Samples were centrifuged at 750 g for 5 min at 4°C and supernatant containing cytoplasm was transferred to a fresh tube. One quarter of the total amount of 5×RIPA buffer [750 mM NaCl (VWR), 5% Igepal CA-630 (Sigma-Aldrich), 2.5% Sodium deoxycholate (Carl Roth), 0.5% SDS (Sigma-Aldrich) and 250 mM Tris (pH 8.0) (AppliChem)] with 1×PI was added. The nuclear pellet was washed with fractionation buffer followed by ten more passes through a 27-gauge needle and 10 min centrifugation at 750 g at 4°C. The supernatant was discarded, the pellet was resuspended in 1×RIPA buffer plus PI and sonicated to shear genomic DNA. Protein isolation was performed according to the procedure described above.
4
Nuclear Fractionation and HAT/HDAC Analysis
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Cells were seeded in 60 mm Petri dishes (SPL Lifescience, Pochon, Republic of Korea) at 3 × 105 per dish and incubated overnight under standard conditions. Afterward, the cells were treated with CBL0137 at concentrations of 0.6 μM and 1.2 μM or DMSO (0.1%) as vehicle control. After 24 h, the cells were washed with PBS and lysed in buffer for nuclear extraction and fractionation (Abcam protocol: 20 mM HEPES pH 7.4 (PanEco, Moscow, Russia), 10 mM KCl (CHIMMED, Moscow, Russia), 2 mM MgCl2 (CHIMMED, Moscow, Russia), 1 mM EDTA (PanEco, Moscow, Russia), 1 mM EGTA (AppliChem, Darmstadt, Germany), 20 mM dithiothreitol (SERVA, Heidelberg, Germany), and Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland)). Briefly, the cells were lysed for 15 min on ice, and then cell suspension was performed through a 26- and 23-gauge needle with the removal of supernatant (for the mechanical destruction of the cytoplasmic and nuclear membrane, respectively). The precipitate was resuspended in deionized water with Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland) and then homogenized with Soniprep 150 Plus ultrasonic homogenizer (MSE, London, UK) for 3 s at a power of 2A on ice. The nuclear fraction was used to analyze the enzymatic activity of HATs as well as the level of HDAC1 and HDAC3 protein expression.
5
Reagents for Neurophysiological Experiments
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The reagents used in the experiments are listed below. (1) Sigma-Aldrich, Saint Louis, MO, United States: Poly(ethyleneimine) solution (Cat. no. P3143), penicillin–streptomycin (Cat. no. P4333), L-Glutamine (Cat. No G85402), Retigabine (Cat. No. SML0325), XE-991 (Cat. no. X2254). (2) Life Technologies, Grand Island, NY, United States: B-27 supplement (Cat. no. 17504044), Trypsin 2.5% (Cat. no. 15090046). (3) Molecular Probes, Eugene, OR, United States: Fura-2 AM (Cat. no. F1221). (4) Cayman Chemical, Ann Arbor, MI, United States: Bicuculline (Cat. no. 11727), 1-naphthylacetyl spermine (NASPM) (Cat. no. 18453). (5) Tocris Bioscience, Bristol, United Kingdom: ATPA (Cat. no. 11–071-0). (6) AppliChem, Darmstadt, Germany: EGTA (Cat. no. A-0878). (7) Dia-M, Moscow, Russian Federation: HEPES (Cat. no. 3350). (8) Paneco, Moscow, Russian Federation: Neurobasal-A medium.
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