The effect of CaM (calmodulin) on [3H]ryanodine binding was assessed as described.37 (link),38 (link) SR-enriched vesicles were obtained from IZ and NIZ, using Tris-buffered solution (0.9% NaCl, 10 mmol/L Tris-HCl, pH 6.8) plus protease inhibitors (PIs) and collected by 3 steps of centrifugation. The 40 000-g pellets were resuspended in Tris-buffered solution with PI, plus 10% sucrose. After protein quantification, the [3H]ryanodine-bind-ing assay was performed. Briefly, 100 μl of [3H]ryanodine-binding buffer containing 200 mmol/L KCl, 25 mmol/L Tris, 50 mmol/L Hepes (pH 7.4), 1 mmol/L EGTA, 5 nmol/L [3H]ryano-dine (68.4 Ci-mmol-1; Dupont NEN), and CaCl2 was added to set free [Ca2+] to pCa 5. Ca2+/EGTA ratio was calculated with Max-Chelator (www.stanford.edu/~cpatton/maxc.html ), and 50 μg of SR-enriched solution was incubated for 2 hours at 37°C, filtered on GF/B glass filters (Whatman) presoaked with water, and washed twice with 5 mL of distilled water with an M24-R cell harvester (Brandel). Nonspecific binding was determined in the presence of 20 μM unlabeled ryanodine and subtracted from each sample.
Gf b glass filters
Manufactured by Cytiva
GF/B glass filters are a type of laboratory filtration equipment. They are designed for the retention of fine particulates during filtration processes. The GF/B filters are made of glass microfibers and provide consistent and reliable performance for a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Whatman, by Cytiva (14 mentions)
Ls6500 liquid scintillation counter, by Beckman Coulter (6 mentions)
Ls6500, by Beckman Coulter (2 mentions)
3h prazosin, by PerkinElmer (2 mentions)
M24r cell harvester, by Brandel Inc (1 mentions)
3h 7 oh dpat, by PerkinElmer (1 mentions)
Ecoscint a, by National Diagnostics (1 mentions)
3h spiperone, by PerkinElmer (1 mentions)
3h 8 oh dpat, by PerkinElmer (1 mentions)
3h mesulergine, by PerkinElmer (1 mentions)
3h rauwolscine, by PerkinElmer (1 mentions)
Prism 9, by GraphPad (1 mentions)
3h paroxetine, by PerkinElmer (1 mentions)
3h mepyramine, by PerkinElmer (1 mentions)
15 protocols using gf b glass filters
1
CaM Regulation of Ryanodine Receptor Binding
2
Rat Olfactory Tubercle Membrane Binding Assay
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Rat olfactory tubercle was homogenized in 20 volumes of ice-cold 50 mM Hepes Na (pH 7.5) using an ULTRA TURAX homogeniser, and centrifuged twice for 10 min at 48,000 g with resuspension of the pellet in fresh buffer. The final pellet was resuspended in 50 mM Hepes Na, pH 7.5, containing 1 mM EDTA, 0.005% ascorbic acid, 0.1% albumin, and 200 nM eliprodil. Total binding each assay tube was added 900 µL of membranes, 50 µL of 0.6 nM [3H] 7-OH-DPAT (50 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL of 50 mM Hepes Na, pH 7.5, containing 1 mM EDTA, 0.005% ascorbic acid, 0.1% albumin, 200 nM eliprodil. Non-specific binding each assay tube was added 900 µL of membranes, 50 µL of [3H] 7-OH-DPAT, 50 µL of 1 µM dopamine. Specific binding each assay tube was added 900 µL of Membranes, 50 µL of [3H] 7-OH-DPAT, 50 µL of 50 µM new compounds. The tubes were incubated at 25 °C for 60 min. The incubation was followed by a rapid vacuum filtration through Whatman GF/B glass filters, and the filtrates were washed twice with 5 mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter.
3
Membrane Fraction Preparation and [35S] GTPγS Binding Assay
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To prepare membrane fractions, cells were harvested in phosphate buffer saline (PBS), frozen at least overnight at −80 °C, and then homogenized in ice-cold 50 mM Tris-HCl buffer, pH 7.5 using a Potter Elvehjem tissue grinder. The nuclear pellet was removed by centrifugation at 1000× g for 15 min at 4 °C. The total membrane fraction was collected after centrifugation of the supernatant at 100,000× g for 35 min at 4 °C. The membrane fraction was aliquoted and stored at −80 °C in 50 mM Tris-HCl, pH 7.4, and the protein concentration was determined by the Bradford method. The [35S] GTPγS binding assays were performed in polypropylene tubes in a buffer consisting of 20 mM HEPES pH 7.4, 100 mM NaCl, 5 mM MgCl2, 0.1% fatty acid-free BSA, and 10−4 M GDP. Membranes were incubated for 60 min at 30°C in the buffer supplemented with 5 µg saponin, 0.2 nM [35S] GTPγS, and 1 mM of the S1PRs agonist FTY720-P. The reaction was stopped by vacuum filtration through Whatman GF/B glass filters preincubated in buffer, which were then washed three times with 4 mL of ice-cold buffer without GDP. Membrane-bound radioactivity was determined by liquid scintillation counting (Packard, GMI Trusted laboratory Solutions, Ramsey, MN, USA) after overnight extraction of the filters in 4 mL of scintillation cocktail (Ecoscint A, National Diagnostics, Fisher Scientific).
4
Rat Striatum Radioligand Binding Assay
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Rat striatum was homogenized in 20 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.7) using an ULTRA TURAX homogeniser, and centrifuged twice for 10 min at 48,000 g with resuspension of the pellet in fresh buffer. The final pellet was resuspended in 50 mM ice-cold Tris-HCl containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% ascorbic acid and 5 µM pargyline. Total binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 0.5 nM [3H]spiperone (16.2 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL Tris-HCl buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% ascorbic acid and 5 µM pargyline. Non-specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]spiperone, 50 µL of 5 µM (+)-butaclamol. Specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]spiperone, 50 µL of 50 µM new compounds. The tubes were incubated at 37 °C for 15 min. The incubation was followed by a rapid vacuum filtration through Whatman GF/B glass filters, and the filtrates were washed twice with 5 mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter.
5
Rat Cortical Receptor Binding Assay
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Rat cerebral cortex was homogenized in 20 volumes of ice-cold Tris-HCl buffer (50 mM, pH 7.7) using an ULTRA TURAX homogeniser, and centrifuged at 32000 g for 20 min. The resulting pellet was resuspended in the same quantity of the buffer centrifuged for 20 min. The final pellet was resuspended in 50 volumes of the Tris-HCl buffer. Total binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 0.6 nM [3H]ketanserine (60.0 Ci/mmol, Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL Tris-HCl buffer. Non-specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]ketanserin, 50 µL of 10 µM methisergide. Specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]ketanserin, 150 µL of 50 µM new compounds. The tubes were incubated at 37 °C for 15 min. The incubation was followed by a rapid vacuum filtration through Whatman GF/B glass filters, and the filtrates were washed twice with 5 mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter.
6
Quantifying preQ0 Incorporation into tRNAGln
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To quantify preQ0 incorporation into tRNAGln a control reaction was run in which [8-14C]-guanine (50 mCi/mmol) was incorporated into the tRNA using M. jannaschii TGT and the tRNA isolated as described above. After quantifying the radiochemical specific activity of the [14C]tRNA, preQ0 was incorporated into the tRNA and aliquots of the reaction taken over time to measure the loss of [8-14C]-guanine (and incorporation of preQ0). The tRNA from each aliquot was precipitated and collected on Whatman GF/B glass filters. The filters were washed with cold ethanol in a vacuum filtration system so as to remove any unbound radioactive material. Once dry, the filters were placed in 7 mL scintillation vials with scintillation cocktail and the radioactivity was measured by scintillation counting.
7
Radioligand Binding Assay for 5-HT1A Receptors
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Rat cerebral cortex was homogenized in 20 volumes of ice-cold Tris-HCl buffer (50 mM, pH 7.7) using an ULTRA TURAX homogeniser, and was then centrifuged at 32000 g for 10 min. The resulting pellet was then resuspended in the same buffer, incubated for 10 min at 37 °C, and centrifuged at 32000 g for 10 min. The final pellet was resuspended in Tris-HCl buffer containing 10 µM Pargyline, 4 mM CaCl2 and 0.1% ascorbic acid. Total binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 0.5 nM [3H]8-OH-DPAT (187.4 Ci/mmol, Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL Tris–HCl buffer containing 10 µM Pargyline, 4 mM CaCl2 and 0.1% ascorbic acid. Non-specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H] 8-OH-DPAT, 50 µL of 10 µM serotonin. Specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]8-OH-DPAT, 50 µL of 50 µM new compounds. The tubes were incubated at 37 °C for 30 min. The incubation was followed by a rapid vacuum filtration through Whatman GF/B glass filters, and the filtrates were washed twice with 5 mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter.
8
Rat Cortex Membrane Binding Assay
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Rat cerebral cortex was homogenized in 20 volumes of ice-cold Tris-HCl buffer (50 mM, pH 7.7) using ULTRA TURAX homogeniser, and centrifuged at 32000 g for 20 min. The resulting pellet was resuspended in the same quantity of the buffer centrifuged for 20 min. The final pellet was resuspended in 50 volumes of the Tris-HCl buffer. Total binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]mesulergine, 50 µL Tris-HCl buffer. Non-specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 1 nM [3H]mesulergine (85.4 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL of 10 µM mianserin. Specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]mesulergine, 50 µL of 50 µM new compounds. The tubes were incubated at 37 °C for 15 min. The incubation was followed by a rapid vacuum filtration through Whatman GF/B glass filters, and the filtrates were washed twice with 5 mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter.
9
Ryanodine Receptor Binding Assay
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HEK293 cells were cultured in five 150 mm dishes, and protein expression was induced with doxycycline (2 μg/ml) for 48 h. Microsomes were prepared by nitrogen cavitation [21 (link)]. The microsomes (50–100 μg of protein) were incubated with 5 nM [3H]ryanodine for 5 h at 25°C in a 100 μl solution containing 0.17 M NaCl, 20 mM 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO), pH 6.8, 2 mM dithiothreitol, 1 mM AMP and various concentrations of free Ca2+ buffered with 10 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). For [3H]ryanodine binding experiments at 37°C, the incubation period was shortened to 3 h. Ca2+ concentrations were calculated using MaxChelator (http://maxchelator.stanford.edu ) [25 (link)]. The protein-bound [3H]ryanodine was separated by filtering through polyethyleneimine-treated Whatman GF/B glass filters. Nonspecific binding was determined in the presence of 20 μM unlabeled ryanodine. The [3H]ryanodine binding data (B) were normalized by the maximum number of functional channels (Bmax), which was separately determined by Scatchard plot analysis using varied concentrations (3–20 nM) of [3H]ryanodine in a high-salt buffer containing 1 M NaCl [8 (link)]. The resultant B/Bmax represents the averaged activity of each mutant.
10
Rat Cortex Binding Assay
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Rat cerebral cortex was homogenized in 20 volumes of ice-cold Tris-HCl buffer containing 5 mM EDTA (50 mM, pH 7.7) using ULTRA TURAX homogeniser, and centrifuged at 44000 g for 20 min at 4°C. The resulting pellet was resuspended in the same quantity of the buffer centrifuged for 20 min. The final pellet was resuspended in 50 volumes of the Tris-HCl buffer. Total binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 1 nM [3H] rauwolscine, 50 µL Tris-HCl buffer. Non-specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 1 nM [3H] rauwolscine (73.0 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL of 10 µM rauwolscine. Specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H] rauwolscine, 50 µL of 50 µM new compounds. The tubes were incubated at 25°C for 60 min. The incubation was followed by a rapid vacuum filtration through Whatman GF/B glass filters, and the filtrates were washed twice with 5 mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter.
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