Radioactive 14C-mannitol and 14C-fructose were purchased from American Radiolabeled Chemicals (St. Louis, Mo). Transport rates were determined as previously described [28, 37, 38 (link)]. Cultures were grown in defined medium containing a carbon of interest to an OD600 between 0.6 and 0.8. The cultures were then washed twice and resuspended to an OD600 of 0.3 in defined salts medium. Assays were initiated by the addition of 14C-mannitol or 14C-fructose to a final concentration of 2 µM. Competing substrates were added to a final concentration of 2 or 10 µM. Subsequently, culture aliquots were passed through a 0.45 µm Hv filter on a Millipore sampling manifold at specified time points. Accumulation of radiolabel was quantified using a liquid scintillation counter (Beckman LS6500). Uptake rates were standardized to total protein.
Sampling manifold
Manufactured by Merck Group
Sourced in United States
The Sampling Manifold is a laboratory equipment designed for the collection and distribution of liquid or gas samples. It provides a controlled and regulated flow of the sample material to multiple sample containers or analysis equipment. The core function of the Sampling Manifold is to facilitate the systematic and consistent sampling process, ensuring accurate and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Ls6500, by Beckman Coulter (4 mentions)
Whatman, by Cytiva (2 mentions)
Disc filters, by Merck Group (2 mentions)
Ultima gold, by PerkinElmer (2 mentions)
14c mannitol, by American Radiolabeled Chemicals (1 mentions)
Gf b filters, by Merck Group (1 mentions)
Tri carb 2100 tr liquid scintillation counter, by Hewlett-Packard (1 mentions)
Ultima gold scintillation fluid, by PerkinElmer (1 mentions)
6 protocols using sampling manifold
1
Radiolabeled Carbohydrate Transport Assays
2
Binding Assay for Opioid Receptors
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Commercial membranes of CHO cells (Perkin Elmer, Inc., Waltham, MA, USA) stably expressing human opioid receptors and the competing radioligands, [3H]DAMGO, [3H]deltorphin-2 and [3H]U-69593 for MOP, DOP and KOP, respectively, were used. Membranes were incubated in 0.5 mL of 50 mM Tris/HCl (pH = 7.4), 0.5% bovine serum albumin (BSA), with a number of peptidase inhibitors (bacitracin, bestatin, captopril) and various concentrations of radioligands for 2 h at 25 °C. Non-specific binding was assessed in the presence of 10 mM naloxone. Assays were incubated at room temperature for 1 h and reactions were terminated via filtration through Whatman GF/B filters, soaked in 0.5% polyethyleneimine (PEI), using Millipore Sampling Manifold (Billerica, MA, USA). Radioactivity was determined after an 8 h extraction period [69 (link)].
3
Monitoring Ribosome E-Site Binding by EttA-EQ2
4
Radioligand Binding Assay for μ-Opioid Receptors
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The μ-opioid receptor-binding studies with the opioid peptide and opioid-BACE1 siRNA were performed according to a previously described method, with some modifications [24 (link)]. Briefly, crude membrane preparations isolated from Wistar rat brains were incubated at 25°C for 120 min with an appropriate concentration of a tested peptide in the presence of 0.5 nM [3H]DAMGO in a total volume of 0.5 mL of 50 mM Tris-HCl (pH 7.4) containing MgCl2 (5 mM), ethylenediaminetetraacetic acid (1 mM), and bacitracin (20 mg/L). Incubation was terminated by rapid filtration through Whatman GF/B (Brentford, UK) glass fiber strips, which had been presoaked for 2 h in 0.5% polyethylamine, using a Sampling Manifold (Millipore, Billerica, MA, USA). The filters were washed 3 times with 4 mL of ice-cold Tris buffer solution. The bound radioactivity was measured with a Tri-Carb 2100 TR liquid scintillation counter (Packard, Ramsey, MN, USA) after overnight extraction of the filters in 4 mL of Ultima Gold scintillation fluid (Perkin-Elmer, Wellesley, MA, USA).
Three independent experiments for each assay were carried out in duplicate.
Three independent experiments for each assay were carried out in duplicate.
5
Radiolabeled Rhamnose Transport Assay
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Transport assays were conducted as previously described [24 (link)]. Radioactive [3H] rhamnose (5 Ci/mmol) was purchased from American Radiolabeled Chemicals Ltd. (St. Louis, MO, USA). Transport assays were initiated by the addition of tritiated rhamnose to a final concentration of 2 µM. Aliquots of 0.5 mL were withdrawn at appropriate time points, and then rapidly filtered through a Millipore 0.45 µm Hv filter on a Millipore sampling manifold. Filtered cells were immediately washed with 5 mL of defined salts medium, and the residual radioactivity on the filter was quantified using a liquid scintillation spectrophotometer (Beckman LS6500). Transport rates were linear over the first minute of the assay.
6
Monitoring Ribosome E-Site Binding by EttA-EQ2
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The assay was based on the original assay of Grajevskaja et al.26 (link) used to discover the E site. The 70S ribosomes used for the assay were prepared as for the minimal in vitro purified translation assay4 . 70S ribosomes (0.2 µM) were incubated in the presence of increasing concentration of EttA-EQ2 (0, 0.2, 0.4, 0.8, 1.0, and 1.2 µM) and deacylated [32P]tRNAPhe (0.4 µM) for 2 min at 4 °C in 0.1 mM ATP, 10 mM Mg(OAc)2, 100 mM NH4Cl, 20 mM Tris-HCl (pH 7.4) in a 20 µl reaction. 5 µl of each reaction were spotted on a nitrocellulose filter (25 mm, 0.45 µm, nitrocellulose, disc filters, Millipore) installed on a Sampling Manifold (Millipore) under constant vacuum. After 3 washes with 2 ml of 20 mM Mg(OAc)2, 100 mM NH4Cl, 1 mM EDTA, 20 mM Tris-HCl (pH 7.4), the filters were immersed in scintillation liquid (Ultima Gold, PerkinElmer) and counted on a scintillation counter (Beckman LS6500). 5 µl of each reaction was also counted without being spotted on a nitrocellulose filter or washed, to give the total radioactivity in each reaction. The ratio of radioactivity retained on the filter versus total were calculated and adjusted to the mole of ribosome in each reaction.
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