Whatman gf b glass fiber filters

The affinity of the synthesized derivatives towards MAO-B was determined in radioligand competition binding assays. The assays were performed using rat brain membrane homogenates and the MAO-B-specific radioligand l-[³H]Deprenyl (obtained from Novandi Chemistry AB, NT1063). Membrane suspension was incubated with 2 nM l-[³H]deprenyl and various concentrations of the test compound in 50 mM Tris−HCl, pH 7.4 buffer containing 120 mM NaCl, 5 mM KCl, 2 mM MgCl₂, at r.t. for 60 min. Non-specific binding was determined in the presence of 10 μM of rasagiline. The reaction was terminated by rapid filtration using Whatman GF/B glass-fiber filters, pre-soaked in 0.3% polyethyleneimine, and a 48-channel harvester (Biomedical Research and Development Laboratories, Gaithersburg, MD, USA) followed by washing four times with ice-cold TRIS-HCl buffer. Filter-bound radioactivity was quantified by liquid scintillation counting. The results represent two single experiments, each performed in triplicate. The determination of the K_D value of L-Deprenyl was acquired with homologous competition with the non-radioactive ligand in the range (10⁻¹¹–10⁻⁵ M). The data were analyzed with GraphPad Prism, Version 4.1 (GraphPad Inc., La Jolla, CA, USA).

Competitive binding assays were conducted on human opioid receptors stably transfected into CHO cells according to the published procedures [60 (link)]. Binding assays were performed using [³H]DAMGO (1 nM), [³H]DPDPE (1 nM), [³H]U69,593 (1 nM) or [³H]nociceptin (0.1 nM) for labeling MOR, DOR, KOR or NOP receptors, respectively. Non-specific binding was determined using 10 µM of the unlabeled counterpart of each radioligand assays were performed in 50 mM Tris-HCl buffer (pH 7.4) in a final volume of 1 mL. In the NOP receptor binding assay, 1 mg/mL BSA was added to the assay buffer. Cell membranes (15–20 µg) were incubated with various concentrations of test compounds and the appropriate radioligand for 60 min at 25 °C. After incubation, reactions were terminated by rapid filtration through Whatman GF/C glass fiber filters. In the NOP receptor binding assay, filtration was made through 0.5% PEI-soaked Whatman GF/B glass fiber 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, USA). Radioactivity retained on the filters was counted by liquid scintillation counting using a Beckman Coulter LS6500 (Beckman Coulter Inc., Fullerton, CA, USA). All experiments were performed in duplicate and repeated three times with independently prepared samples.

Membranes were prepared from Sprague-Dawley rat or guinea pig brains as previously described [32] (link). All binding experiments were performed in 50 mM Tris-HCl buffer (pH 7.4) in a final volume of 1 ml containing 300–500 µg protein [32] (link). Rat brain membranes were incubated either with [³H]DAMGO (1 nM, 45 min, 35°C) or [³H][Ile^5,6]deltorphin II (0.5 nM, 45 min, 35°C). Guinea pig brain membranes were incubated with [³H]U69,593 (1 nM, 30 min, 30°C). Nonspecific binding was determined in the presence of 10 µM naloxone. Reactions were terminated by rapid filtration using a Brandel Cell Harvester (Brandel Inc., Gaithersburg, MD) and Whatman GF/B glass fiber filters pre-soaked in 0.1% polyethylenimine for 1 h at 4°C for [³H]U69,593, or type GF/C for [³H]DAMGO and [³H][Ile^5,6]deltorphin II. Filters were washed three times with 5 ml of ice-cold 50 mM Tris-HCl buffer (pH 7.4) and bound radioactivity was measured by liquid scintillation counting. All experiments were performed in duplicate and repeated at least three times. Protein concentration was determined by the Bradford method using bovine serum albumin as the standard [33] (link).

Saturation binding experiments at A₃AR were performed using [³H]-5N-(4-methoxyphenyl-carbamoyl) amino-8-propyl-2-(2-furyl) pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidine ([3H]-MRE 3008F20, specific activity 67 Ci/mmol; GE Healthcare) as a radioligand (19 (link)). Membranes (80 μg of protein/assay) were incubated with [3H]-MRE 3008F20 (0.1–30 nM) at 4°C for 150 min. Nonspecific binding was determined in the presence of MRE 3008F20 (1 μM). At the end of the incubation time, bound and free radioactivity were separated by filtering the assay mixture through Whatman GF/B glass fiber filters (Whatman, Maidstone, UK) by using a Brandel cell harvester (Brandel Instruments, Gaithersburg, MD, USA). The filter-bound radioactivity was counted using a Perkin Elmer 2810 TR liquid scintillation counter (Perkin Elmer).

Binding of [³⁵S]GTPγS to membranes from CHO cells stably expressing the human MOR(CHO-hMOR) was conducted according to the published procedure [43 (link)]. Cell membranes (5-10 µg) in 20 mM HEPES, 10 mM MgCl₂, and 100 mM NaCl, pH 7.4 were incubated with 0.05 nM [³⁵S]GTPγS, 10 µM GDP and various concentrations of test peptides in a final volume of 1 mL, for 60 min at 25°C. Non-specific binding was determined using 10 µM GTPγS, and the basal binding was determined in the absence of test ligand. Samples are filtered over glass Whatman glass GF/B fiber filters and counted as described for binding assays. In each individual experiment, the increase in [³⁵S]GTPγS binding produced by the test peptides were normalized to the maximal stimulation of the reference full MOR agonist, DAMGO and nonlinear regression performed on each individual curve were averaged to yield potency (EC₅₀, in nM) and efficacy (as % stim.) values. All experiments were performed in duplicate and repeated at least three times.