GTPγS binding was measured using a filter binding method as described previously (Garcia-Marcos et al., 2010 (link), 2011b (link)). His-Gαi3 (100 nM) was preincubated with different concentrations of His-Daple-CT (aa 1650–2028) for 15 min at 30°C in assay buffer (20 mM Na-HEPES, pH 8, 100 mM NaCl, 1 mM EDTA, 25 mM MgCl2, 1 mM DTT, 0.05% [wt:vol] C12E10). Reactions were initiated at 30°C by adding an equal volume of assay buffer containing 1 µM [35S] GTPγS (∼50 c.p.m/fmol). Duplicate aliquots (25 μl) were removed at different time points, and binding of radioactive nucleotide was stopped by addition of 3 ml ice-cold wash buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 25 mm MgCl2). The quenched reactions were rapidly passed through BA-85 nitrocellulose filters (GE Healthcare) and washed with 4 ml wash buffer. Filters were dried and subjected to liquid scintillation counting. To determine the specific nucleotide binding, the background [35S] GTPγS detected in the absence of G protein was subtracted from each reaction and data expressed as percentage of the [35S] GTPγS bound by His-Gαi3 in the absence of His-Daple-CT.
Ba 85 nitrocellulose filters
Manufactured by GE Healthcare
The BA-85 nitrocellulose filters are a type of laboratory filtration equipment used for various applications. These filters are made of nitrocellulose material and are designed to effectively separate and retain particles, molecules, or cells from a liquid or gas solution. The BA-85 filters are available in a range of pore sizes to accommodate different filtration requirements. They are commonly used in various research and diagnostic procedures where precise filtration is necessary.
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Lab products found in correlation
3 protocols using ba 85 nitrocellulose filters
1
GTPγS Binding Assay for Daple-Gαi Interaction
2
GTPγS Binding Assay for Gαi3
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His-Gαi3 (100 nM) was preincubated with different concentrations of His-xDAPLE-CT (aa 1,638–1,932) for 15 min at 30°C in assay buffer (20 mM Na-Hepes, pH 8, 100 mM NaCl, 1 mM EDTA, 25 mM MgCl2, 1 mM DTT, and 0.05% [wt/vol] C12E10]. Reactions were initiated at 30°C by adding an equal volume of assay buffer containing 1 µM [35S] GTPγS (∼50 cpm/fmol). Duplicate aliquots (25 µl) were removed at different time points, and binding of radioactive nucleotide was stopped by addition of 3 ml ice-cold wash buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, and 25 mm MgCl2). The quenched reactions were rapidly passed through BA-85 nitrocellulose filters (GE Healthcare) and washed with 4 ml cold wash buffer. Filters were dried and subjected to liquid scintillation counting. Background [35S]GTPγS detected at 15 min in the absence of G protein was subtracted from each reaction and data expressed as percentage of the [35S]GTPγS produced by His-Gαi3 in the absence of xDAPLE. Background counts were <5% of the counts detected in the presence of G proteins.
3
GTPγS Binding to Purified His-Gαi3
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GTPγS binding to purified His-Gαi3 was determined as described previously (Garcia-Marcos et al., 2010 (link); Leyme et al., 2014 (link)). Purified His-Gαi3 (100 nM) was diluted in assay buffer (20 mM Na-HEPES, pH 8, 100 mM NaCl, 1 mM EDTA, 25 mM MgCl2, 1 mM DTT, 0.05% (wt:vol) C12E10) and pre-incubated with purified GST-LOV2GIVe proteins (2 µM final) for 15 min at 30°C. Reactions were initiated, by adding an equal volume of assay buffer containing 1 µM [35S]GTPγS (~50 c.p.m/ fmol) at 30°C. Duplicate aliquots (25 μL) were removed 15 min after the reaction start, and binding of radioactive nucleotide was stopped by addition of 2 mL of ice-cold wash buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 25 mM MgCl2). The quenched reactions were rapidly passed through BA-85 nitrocellulose filters (GE Healthcare) and washed with 2 mL cold wash buffer. Filters were dried and subjected to liquid scintillation counting. Background [35S]GTPγS detected in the absence of G-protein was subtracted from each reaction and data expressed as percentage of the [35S]GTPγS bound to His-Gαi3 in the absence of GST-LOV2GIVe.
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