Striata (inclusive of the dorsal and ventral regions) from adult male mice were dissected and homogenized by tissue tearer and glass homogenizer in homogenization buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM EDTA, 1mM DTT), passed through a 26 gauge needle 8 times, centrifuged twice at 20,000 g for 30 minutes at 4°C, and resuspended in assay buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA and 20 μM GDP, 1mM DTT). For each reaction, 2.5 μg of membrane protein were incubated in assay buffer containing ~ 0.1 nM of [35S]GTPγS and increasing concentrations of compounds in a total volume of 200 μL for 2 hours at room temperature. The reactions were terminated by separating membrane bound and free [35S]GTPγS through filtration with GF/B filters using a 96-well plate harvester (Brandel Inc., Gaithersburg, MD). Filters were dried overnight and radioactivity was determined with a TopCount NXT HTS microplate scintillation and luminescence counter (PerkinElmer). Mice were used between the ages of 4–7 months; no apparent changes in receptor coupling were noted in mice from different ages within this range.
96 well plate harvester
Manufactured by Brandel Inc
Sourced in United States
The 96-well plate harvester is a laboratory equipment used for the automated harvesting of cell cultures or other biological samples from 96-well microplates. It is designed to efficiently and consistently transfer samples from the wells to a collection device, such as a filter plate or a tube rack, for further processing or analysis.
Automatically generated - may contain errors
3 protocols using 96 well plate harvester
1
GTPγS Binding Assay in Mouse Brain
2
Ryanodine Receptor Binding Assay
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Binding reactions were set up in a 96 well flat bottom microtiter plate in a total volume of 250 μl per well. Reactions consisted of either 25 μg of membrane prep, [3H] ryanodine (concentrations 0.01–40 nM) and 2 μl of DMSO in a binding buffer containing 0.01% Pluronic (1.5M KCl, 10 mM ATP, 1.38 mM CaCl2, 10 mM HEPES pH 7.4) or 25 μg of membrane prep, [3H] ryanodine (0.01–40 nM), 2 μl of ryanodine in DMSO (final concentration 10 μM) in 0.01% Pluronic. The reactions were incubated at room temperature for 2 h. The 96 samples were then loaded onto a 96 well filter plate pre-treated with 50 μl of 0.1% polyethylenimine, using a 96 well plate harvester (Brandel, MD, USA). Each filter with bound membranes was then washed 3 times with 250 μl of wash buffer (150 mM KCl, 10 mM HEPES pH 7.4 and left overnight at room temperature to dry. Each well on the plate was then filled with 50 μl MicroScint™-O (PerkinElmer, MA, USA) and the filter plate loaded into a TopCount NXT™ Micro-plate Scintillation counter (PerkinElmer, MA, USA). Specific binding and binding kinetics values (equilibrium dissociation constant Kd and Binding capacity/receptor density Bmax) were calculated using GraphPad Prism v5.5 (GraphPad, CA, USA).
3
Membrane-Based Receptor Binding Assay
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Cell membranes were prepared from
CHO cells expressing rat M4 receptors cotransfected with
Gqi5. Cells were harvested, collected by centrifugation,
resuspended in ice-cold homogenization buffer (50 mM Tris-HCl, 0.9%
NaCl, pH 7.4), and then homogenized by 3 × 10 s bursts with a
homogenizer. Cell fractions were separated by centrifugation, and
the resulting pellet was resuspended in ice-cold assay buffer (100
mM NaCl, 10 mM MgCl2, 20 mM HEPES, and 10 mM EDTA, pH 7.4).
For inhibition binding experiments, membranes (10 μg/well) were
incubated with 300 pM [3H]NMS, a fixed concentration of
M4 PAM (300 nM to 10 μM) or vehicle, and a range
of concentrations of ACh (1 nM to 1 mM) for 3 h at room temperature
with shaking in assay buffer. Nonspecific binding was determined using
10 μM atropine. Assays were terminated by rapid filtration using
a Brandel 96-well plate harvester and washed three times with ice-cold
assay buffer. The next day MicroScint20 was added, and radioactivity
was counted. Data (mean ± SEM, n ≥ 2)
were analyzed using GraphPad Prism 5.04.
CHO cells expressing rat M4 receptors cotransfected with
Gqi5. Cells were harvested, collected by centrifugation,
resuspended in ice-cold homogenization buffer (50 mM Tris-HCl, 0.9%
NaCl, pH 7.4), and then homogenized by 3 × 10 s bursts with a
homogenizer. Cell fractions were separated by centrifugation, and
the resulting pellet was resuspended in ice-cold assay buffer (100
mM NaCl, 10 mM MgCl2, 20 mM HEPES, and 10 mM EDTA, pH 7.4).
For inhibition binding experiments, membranes (10 μg/well) were
incubated with 300 pM [3H]NMS, a fixed concentration of
M4 PAM (300 nM to 10 μM) or vehicle, and a range
of concentrations of ACh (1 nM to 1 mM) for 3 h at room temperature
with shaking in assay buffer. Nonspecific binding was determined using
10 μM atropine. Assays were terminated by rapid filtration using
a Brandel 96-well plate harvester and washed three times with ice-cold
assay buffer. The next day MicroScint20 was added, and radioactivity
was counted. Data (mean ± SEM, n ≥ 2)
were analyzed using GraphPad Prism 5.04.
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