The solid-phase binding assay was performed in FluoroNunc Maxisorb 96 well plates (Nunc). The wells were incubated overnight at 4 °C with 200 μl of various proteins at 50 μg/ml dissolved in coating buffer (pH 9.6) containing 15 mM Na2CO3, 35 mM NaCO3 and 0.02% NaN3. The wells were washed three times with 300 μl of assay buffer (pH 7.6), containing 10 mM Tris, 0.14 M NaCl, 0.5 mM CaCl2, 0.02% NaN3 and 0.05% Tween20. The recombinant N-terminal domain of STIM1 was fluorescently labeled, using the EZ-Label Fluorescein Protein Labeling Kit from Pierce. One hundred microliters of varying concentrations of labeled protein were incubated overnight in the wells at 4 °C. The wells were washed twice with assay buffer and a plate reader was used to read the fluorescence. Known amounts of labeled STIM1 were included in each assay to construct a standard curve. Wells were coated with BSA as a negative control.
Fluoronunc maxisorb 96 well plates
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
The FluoroNunc Maxisorb 96 well plates are a high-quality laboratory product designed for a variety of applications. These plates feature a Maxisorp surface, which provides a high-binding capacity for efficient immobilization of biomolecules. The plates are suitable for use in applications such as ELISA, cell-based assays, and other fluorescence-based experiments.
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Lab products found in correlation
3 protocols using fluoronunc maxisorb 96 well plates
1
Solid-Phase Binding Assay for STIM1
2
Solid-Phase Binding Assay for STIM1
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The solid-phase binding assay was performed in FluoroNunc Maxisorb 96 well plates (Nunc). The wells were incubated overnight at 4 °C with 200 μl of various proteins at 50 μg/ml dissolved in coating buffer (pH 9.6) containing 15 mM Na2CO3, 35 mM NaCO3 and 0.02% NaN3. The wells were washed three times with 300 μl of assay buffer (pH 7.6), containing 10 mM Tris, 0.14 M NaCl, 0.5 mM CaCl2, 0.02% NaN3 and 0.05% Tween20. The recombinant N-terminal domain of STIM1 was fluorescently labeled, using the EZ-Label Fluorescein Protein Labeling Kit from Pierce. One hundred microliters of varying concentrations of labeled protein were incubated overnight in the wells at 4 °C. The wells were washed twice with assay buffer and a plate reader was used to read the fluorescence. Known amounts of labeled STIM1 were included in each assay to construct a standard curve. Wells were coated with BSA as a negative control.
3
DPP Activity Assay of Tumor Cell Proteins
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The DPP activity of these protein products produced by tumor cells was performed as previously described33 (link). Briefly, an immunocapture assay was performed using Ala-Pro-7-amido-4- trifluoromethylcoumarin (Ala-Pro-AFC) as a substrate (Bachem, Torrance, CA, USA). Fluoronunc MaxiSorb 96-well plates (Nunc, Roskilde, Denmark) were coated overnight at 4 °C with 100 μg/mL anti-FAP antibodies and washed with phosphate-buffered saline containing 0.1% Tween20 (PBST), blocked in 5% bovine serum albumin for 1 h at room temperature. Total protein from pFAP-transfected, pVector-transfected and untransfected tumor cells was extracted using detergent protein extraction reagent (Pierce Biotechnology, Rockford, IL, USA), according to the manufacturer’s instructions. The protein concentration was measured using the Lowry method. Approximately 1 mg of total protein extracts was added to each well and incubated for 1 h, followed by 10 washes with PBST. The DPP activity was assessed by cleavage of 0.25 mmol/L Ala-Pro-AFC for 1 h at room temperature. Release of the free AFC fluorescent substrate was detected using a cytofluor fluorimeter (Labsystems, Helsinki, Finland) with 396 nm excitation and 490 nm emission wave lengths.
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