Binding affinities (IC50) for the compounds under study were determined by a competitive cell-binding assay with PSMA-positive LNCaP cells, using known protocols.60 ,61 On a 96-well plate (MultiScreen HTS-DV 0.65 μm) LNCaP cells (105 per well) were incubated with 0.75 nM [68Ga]Ga-HBED-CC-[Glu-urea-Lys(Ahx)]2 (68Ga-PSMA-10) (16–20 MBq/nmol) in the presence of 12 different concentrations of PSMA PLT-TM or PSMA-11 ranging from 0 − 5000 nM (100 μL/well). After incubation at RT for 45 min with gentle agitation, the binding buffer was removed using a multiscreen vacuum manifold (Millipore, Billerica, MA). After washing twice with 100 μL and once with 200 μL ice-cold binding buffer, the cell-bound radioactivity was measured with a gamma counter. The 50% inhibitory concentration (IC50) and values were calculated by fitting the data using a nonlinear regression algorithm (GraphPad Software, La Jolla, CA, USA). Experiments were performed in quintuplicate.
Multiscreen vacuum manifold
Manufactured by Merck Group
Sourced in United States, Germany
The Multiscreen vacuum manifold is a laboratory equipment used for efficient filtration and separation of liquid samples. It provides a controlled vacuum environment to facilitate the simultaneous processing of multiple samples. The manifold allows for consistent and reliable sample preparation across various analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Multiscreen separation system, by Merck Group (1 mentions)
Falcon tube, by BD (1 mentions)
Thermomax microtitre plate reader, by Molecular Devices (1 mentions)
Multiscreen 96 well filter plate, by Merck Group (1 mentions)
Immobilon p, by Merck Group (1 mentions)
2470 wizard2, by PerkinElmer (1 mentions)
7 protocols using multiscreen vacuum manifold
1
PSMA Binding Affinity Assay
2
FMRP RNA Binding Assay
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RNA binding assay was performed as previously described (6 (link),18 (link)–19 ) using a filter binding assay using Multiscreen Vacuum Manifold and the Multiscreen Separation System (Millipore). A total of 2 pmol of recombinant FMRP were used in each reaction.
3
Irradiation-induced dNTP pool changes
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L1210-10K cells ± dCK (WT, S74A, S74E) were irradiated using a Cs-137 source at a rate of 7.16 Gy/min to a total dose of 3 Gy and kept in 15 ml Falcon tubes (BD Biosciences) at 37°C, 5% CO2. Each hour after irradiation, 1×106 cells were taken out, washed once with PBS, pelleted and re-suspended in 500 µl of ice-cold 60% methanol. Cells were vortexed for 1 min and stored at −20°C overnight. Next day lysates were exposed to 95°C for 3 minutes, and pelleted at 17,000×g for 15 minutes. Supernatants were evaporated for 4 hours in Speed VacPlus SC110A (Savant), and pellets were re-suspended in 100 µl of nuclease-free H2O. Previously described protocol was used to measure dNTP pools [44] (link), with the following modifications. Reactions were carried out simultaneously in triplicate in 96-well U-bottom plates (Becton Dickinson) using 5 µl of lysate in 25 µl total volume per well. After incubation for 2 hours at 37°C, 20 µl from each well were transferred to 96-well DE81 Unifilter plates (Whatman). Plates were washed 4 times with Na2HPO4, once with diH2O and once with 95% ethanol using Multiscreen vacuum manifold (Millipore). Washed plates were dried, and 100 µl of scintillation fluid was added to each well. Radioactivity was measured with a BetaMax plate reader (PerkinElmer).
4
Screening of Resin-Bound Peptides for Antibodies
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Screening of resin-bound peptides was conducted as previously described [25 (link)]. Briefly, resin-bound peptides were added to a 96-well multiscreen filterplate (Millipore, Copenhagen, Denmark) and rinsed with TTN buffer (3 x 1 min), followed by blocking in TTN for 20 min. All incubations with antibodies diluted in TTN were carried out for 1 h at RT followed by three washes in TTN buffer. The resin beads were washed using a multiscreen vacuum manifold (Millipore, Billerica, MA, USA). Human patient sera and healthy control sera were used as primary antibody (200-fold dilution), while AP-conjugated goat anti-human IgG was used as secondary antibody (1 μg/mL). Bound antibodies were quantified using pNPP (1 mg/mL) diluted in AP substrate buffer. Finally, the buffer was transferred to a Maxisorp microtitre plate (Nunc, Roskilde, Denmark) and the absorbance was measured at 405 nm, with background subtraction at 650 nm, on a Thermomax microtitre plate reader (Molecular Devices, Menlo Park, CA, USA).
5
Automated Proteomics Sample Preparation
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A liquid handling robotic system (Agilent Technologies, Santa Clara, CA, USA) controlled by VWorks software was used to perform the automated FASP [87 (link),88 (link)]. Multiscreen Vacuum Manifold™ (Millipore, Billerica, MA, USA) was used to supply vacuum during sample preparation. In brief, protein samples were first loaded onto 50 wells of a 96-well plate (AcroPrep Advance Filter Plates, 350 µL, Omega 30K MWCO) with 100 µg protein/well. Proteins were reduced with 100 µL of 5 mM TCEP (37 °C, 30 min), alkylated with 100 µL of 50 mM IAA (25 °C, 30 min, in the dark), and digested with trypsin at an enzyme: protein ratio of 1:50 (w/w) (37 °C, 18 h). The digestion was stopped by reducing the pH of the samples to 2–3 with formic acid. The number of samples was reduced from 50 to 5 by pooling before desalting.
6
Recombinant Mouse CD31/PECAM-1 Protein Binding Assay
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Recombinant Mouse CD31/PECAM-1 Protein (extracellular domain of muPECAM-1 with predicted molecular mass of 65 kDa) was coated on 96-well Multiscreen Filter Plate, Immobilon P (EMD Millipore, Darmstadt, Germany) at a concentration of 5 μg/mL for 2 h at 37°C. Subsequent steps of washing, blocking as well as RIA for the assessment of binding parameters and data analysis were performed as described above for cellular homogenates using vacuum filtration with MultiScreen Vacuum Manifold (EMD Millipore, Darmstadt, Germany).
7
Quantifying Antibody Binding Kinetics
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Live-cell cellular homogenates were coated on 96-well Multiscreen Filter Plate, Immobilon P (EMD Millipore, Darmstadt, Germany) at a concentration of 0.67 mg/mL (100 μL each well) for 2 h at 37°C. Wells were triple washed with washing buffer (10% (v/v) FBS in PBS) using vacuum filtration with MultiScreen Vacuum Manifold (EMD Millipore, Darmstadt, Germany). To block non-specific binding, wells were incubated with blocking buffer (1% (w/v) BSA in PBS) for 2 h at 37°C with subsequent triple washes as described. Wells were then incubated with increasing concentrations of [125I]-mAb (1.6 pM–20 nM in assay buffer) “solo” or “paired” with fixed concentration of unlabeled mAb (25 nM) in triplicates at RT for 2 h at RT with mild shaking of the plate. To stop binding reaction, cells were filter-washed four times with ice-cold washing buffer. Non-specific binding (NSB) was calculated by measuring radiolabeled ligand binding to wells coated with 3% (w/v) BSA in PBS. Experimental data was measured by a gamma counter (Wizard2 2470, Perkin Elmer, MA), analysis was performed as described for live-cell RIA.
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