Proteoliposomes were prepared for transport assays by extruding them through a 400 nm filter 11 times and concentrating them to 80 mg/ml lipid using ultracentrifugation. The transport assays were started by adding the proteoliposomes to Reaction Buffer (20 mM Tris/HEPES, pH 7.5, 100 mM NaCl, 100 mM KCl, 1 μM valinomycin, 1 μM [3H]-succinic acid (American Radiolabeled Chemicals). At the indicated times, samples were taken and the reaction was terminated by adding the sample to ice cold Quench buffer (20 mM Tris/HEPES, pH 7.5, 200 mM ChCl) and rapidly filtering the sample through a 200 nm nitrocellulose filter (Millipore). The filter was washed with 3 ml Quench buffer, the filters were dissolved in FilterCount liquid scintillation cocktail (PerkinElmer) and the [3H]-succinic acid internalized by the proteoliposomes was counted using a Trilux beta counter (PerkinElmer).
Trilux beta counter
Manufactured by PerkinElmer
The Trilux beta counter is a laboratory instrument designed to measure and quantify beta radiation. It utilizes a scintillation counting technique to detect and analyze the presence of beta-emitting samples. The Trilux beta counter provides accurate and reliable measurements for various applications in fields such as research, environmental monitoring, and radiochemical analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose filter, by Merck Group (3 mentions)
Valinomycin, by American Radiolabeled Chemicals (2 mentions)
3h succinic acid, by American Radiolabeled Chemicals (2 mentions)
Filtercount liquid scintillation cocktail, by PerkinElmer (2 mentions)
3h succinic acid, by PerkinElmer (2 mentions)
Optiphase hisafe 3, by PerkinElmer (1 mentions)
L 3h amino acids, by PerkinElmer (1 mentions)
5 protocols using trilux beta counter
1
Proteoliposome Transport Assay Protocol
2
Transporter Function Assay in Oocytes
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Oocytes were injected with RNA-encoding transporters or an equivalent volume of water as a negative control. A minimum of 3 days after injection, oocytes were incubated for 10 min in uptake solutions containing 3H-l -substrates (PerkinElmer) ± GPNA where specified, in ND96. Oocytes were subsequently rinsed three times in ice-cold ND96. Cells were then lysed in 1 M NaOH and 1% SDS. 3H-l -substrate uptake was measured by scintillation counting using OptiPhase HiSafe 3 (PerkinElmer) and a Trilux beta counter (PerkinElmer). Raw data from each oocyte were normalized to the mean response from the corresponding positive control (all normalized datapoints shown). Inhibition–response data generated using uptake were fit as detailed previously.
3
Radiolabeled Amino Acid Uptake Assay
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Uptake of L-[3H]amino acids (PerkinElmer Life Sciences) was measured in oocytes expressing wild-type and mutant ASCT1, and uninjected oocytes. Five oocytes were incubated in Cl−-containing buffer with 10 µM L-[3H]amino acids at room temperature. After 10 min, uptake was terminated by three rapid washes in ice-cold Cl−-containing buffer followed by lysis in 50 mM NaOH and 1% SDS. L-[3H]amino acids were measured by scintillation counting using a Trilux beta counter (PerkinElmer Life Sciences).
4
Radiolabeled Substrate Transport Assay
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Mutant and wild-type GltPh-mediated transport of radiolabelled substrate was assayed by diluting proteoliposomes (100 mg lipid per ml) 133-fold into uptake buffer (100 mM NaCl, 20 mM HEPES-Tris pH 7.5, 1 μM valinomycin, 100 nM–1 µM L-[3H]substrate) pre-warmed to 30 °C. At each time point, a 200 µl aliquot was removed and diluted 10-fold into ice-cold quench buffer (100 mM LiCl, 20 mM HEPES-Tris pH 7.5), followed by immediate filtration over nitrocellulose filters (0.22 μm pore size, Millipore). The filters were washed with ice-cold quench buffer and assayed for radioactivity using a Trilux beta counter (PerkinElmer). To establish substrate concentration–response curves, extraliposomal L-[3H]substrate was varied between 500 nM and 100 µM, maintaining NaCl concentrations at 300 mM. For inhibitor concentration–response curves, for wild-type GltPh 100 nM L-[3H]aspartate, and for mutant GltPh 1 µM L-[3H]serine, was co-applied with varying inhibitor concentrations. Background levels of uptake were measured by diluting proteoliposomes into internal buffer (100 mM KCl, 20 mM HEPES-Tris pH 7.5) containing 1 µM of valinomycin and the indicated concentrations of L-[3H]substrate.
5
Proteoliposome Transport Assay Protocol
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