Translation of poly(U) templates was carried out as described (Synetos, Frantziou and Alksne 1996 (link)) using 5 A260 units of ribosomes, 25 mg poly(U), 100 mg of soluble protein factors, 25 mg deacylated yeast tRNA and 0,3 nmol [3H]-phenylalanine. The reaction was performed at 30°C for 30 min. The products were precipitated in 10% TCA, and the ester bond between the tRNAs and poly(Phe) peptides was disrupted at 95°C for 10 min, followed by cooling on ice for 10 min in order to release precipitated polypeptide chains. Then the filtration through the Whatmann glass fiber GF/C filters was performed. Filters were washed with 1 mL of 5% TCA and 1 mL of 95% ethanol and subjected to scintillation counting. The in vitro translation assays were performed in triplicate. The values reported are corrected for the control samples lacking ribosomes, which were typically 1%–2% of the total counts of a probe applied.
Glass fiber gf c filters
Manufactured by Cytiva
Glass fiber GF/C filters are laboratory filtration products designed to separate and retain particulates from liquid samples. They are made of borosilicate glass fibers and provide a consistent, high-quality filtration medium for a variety of applications.
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Lab products found in correlation
2 protocols using glass fiber gf c filters
1
In Vitro Poly(Phe) Translation Assay
2
Opioid Receptor Binding Assays
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Binding
assays were performed in 50 mM Tris-HCl buffer (pH 7.4) in a final
volume of 1 mL, with rodent brain preparations (0.3–0.5 mg
protein) or membranes from CHO cells expressing the human opioid receptors
(15–20 μg) and various concentrations of test compound
as described previously.44 (link),49 (link),64 (link) Rat brain membranes were incubated with either [3H]DAMGO
(1 nM, 45 min, 35 °C) or [3H][Ile5,6]deltorphin
II (0.5 nM, 45 min, 35 °C) for labeling MOR or DOR receptors,
respectively. Guinea-pig brain membranes were incubated with [3H]U69,593 (1 nM, 30 min, 30 °C) for labeling KOR. Binding
assays with CHO cell membranes were conducted at 25 °C for 60
min using [3H]DAMGO (1 nM) or [3H]diprenorphine
(0.2 nM) for labeling MOR or DOR, respectively. Nonspecific binding
was determined using 10 μM naloxone (rodent brain) or 1–10
μM of the unlabeled counterpart of each radioligand (CHO cells).
Reactions were terminated by rapid filtration through Whatman glass
fiber GF/C filters. Filters were washed three times with 5 mL of ice-cold
50 mM Tris-HCl buffer (pH 7.4) using a Brandel M24R cell harvester
(Gaithersburg, MD). Radioactivity retained on the filters was counted
by liquid scintillation counting using a Beckman Coulter LS6500 (Beckman
Coulter Inc., Fullerton, CA). All binding experiments were performed
in duplicate and repeated at least three times.
assays were performed in 50 mM Tris-HCl buffer (pH 7.4) in a final
volume of 1 mL, with rodent brain preparations (0.3–0.5 mg
protein) or membranes from CHO cells expressing the human opioid receptors
(15–20 μg) and various concentrations of test compound
as described previously.44 (link),49 (link),64 (link) Rat brain membranes were incubated with either [3H]DAMGO
(1 nM, 45 min, 35 °C) or [3H][Ile5,6]deltorphin
II (0.5 nM, 45 min, 35 °C) for labeling MOR or DOR receptors,
respectively. Guinea-pig brain membranes were incubated with [3H]U69,593 (1 nM, 30 min, 30 °C) for labeling KOR. Binding
assays with CHO cell membranes were conducted at 25 °C for 60
min using [3H]DAMGO (1 nM) or [3H]diprenorphine
(0.2 nM) for labeling MOR or DOR, respectively. Nonspecific binding
was determined using 10 μM naloxone (rodent brain) or 1–10
μM of the unlabeled counterpart of each radioligand (CHO cells).
Reactions were terminated by rapid filtration through Whatman glass
fiber GF/C filters. Filters were washed three times with 5 mL of ice-cold
50 mM Tris-HCl buffer (pH 7.4) using a Brandel M24R cell harvester
(Gaithersburg, MD). Radioactivity retained on the filters was counted
by liquid scintillation counting using a Beckman Coulter LS6500 (Beckman
Coulter Inc., Fullerton, CA). All binding experiments were performed
in duplicate and repeated at least three times.
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