The BRET was done in a 384-well plate, with each well having a total of 50 μL of total reaction volume consisting of 1 nM (binding sites) purified ion channel tagged with Nluc, 500 nM BODIPY-TMR phosphatidylinositol 4,5-bisphosphate (FL-PIP2) (Echelon Biosciences), various concentrations of competing ligand, and 1:2,000 furimazine (NanoGlo; Promega), the substrate for Nluc, in the SEC buffer with 7 mM DDM final concentration. BRET signal brought about by the binding of FL-PIP2 to TREK-1-Nluc and competition with a ligand was calculated by subtracting signal from Nluc-only control enzyme (purified from bacteria) and measured using Envision Multilabel 2104 plate reader (PerkinElmer) set for dual-emission detection (540 nm for Nluc and 574 nm for FL-PIP2) and automatic BRET ratio calculation. Nluc control enzyme was purified from BL1 bacteria cells. Although not used here, background fluorescence can be determined using excess non-FL-PIP2 (e.g., C8-PIP2) rather than the Nluc control enzyme. Data points were fit to a sigmoidal dose-response curve with variable slope using GraphPad Prism. A FRET version of the assay employed a GFP tag rather than an Nluc tag. See also Figure S2A and Supplemental Experimental Procedures .
Envision 2104 multilabel
Manufactured by PerkinElmer
Sourced in United States
The EnVision 2104 multilabel is a high-performance microplate reader designed for a wide range of applications in life science research and drug discovery. It offers accurate and sensitive detection of various assays, including fluorescence, luminescence, and absorbance measurements.
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Lab products found in correlation
5 protocols using envision 2104 multilabel
1
BRET-Based PIP2 Binding Assay for Ion Channels
2
BRET-Based PIP2 Binding Assay for Ion Channels
3
Protein Harvesting and Quantification Protocol
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Following treatment, the protein was harvested, as previously described [33 (link),39 (link)], using a lysis buffer of composition 1× RIPA buffer (Cat # R0278 Sigma-Aldrich, St. Louis, MO, USA), containing 10 μL/mL of Halt protease and phosphatase inhibitor (Cat # 78440, Thermo-Fisher Scientific, Waltham, MA, USA) and 10 μL/mL of 0.5 M EDTA pH = 8.0 (Cat # 15575020, Thermo-Fisher Scientific, Waltham, MA, USA). The cell extracts were lysed 8–10 times through a 23 g needle on a 3 mL syringe maintained on ice for 30–45 min with intermittent vortexing and was then centrifuged at 13,000 rpm for 10 min at 4 °C using the Eppendorf 5417R refrigerated centrifuge (Eppendorf, Hauppauge, NY, USA). The supernatant (protein lysate) was used for immunoblotting. The total protein concentration of each of the samples was estimated as per kit protocol using the Bio-Rad DC protein assay kit (Cat # 5000112, Bio-Rad, Hercules, CA, USA). The absorbance was then read at 750 nm using a 96-well ‘EnVision 2104 multilabel’ plate reader (PerkinElmer, Inc., Waltham, MA, USA).
4
Kinase Inhibition Profiling Assays
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In vitro enzymatic activity was measured using Mobility Shift Assay, Lance Ultra Assay, Lance Screen Assay, Kinases-Glo Assay and ADP-Glo Assay. Kinases were purchased from BPS, Carna, Invitrogen, Millipore and ProQinase. Kinases were seeded in 384-well plates and treated with ten different concentrations of each compound (ranging between 0.05 nM and 10 µM) in duplicate. Staurosporine was used as positive control. Fluorescence polarization were read by Envision 2104 Multilabel (PERKINELMER INC). The 50% inhibitory concentration (IC50) was calculated by fitting the dose-response curve to a 4-parameter log-logistic equation respectively.
5
Quantifying Lysostaphin Levels via ELISA
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Levels of lysostaphin were determined using enzyme-linked immunosorbent assay (ELISA). 96-well EIA/RIA plates were coated overnight at 4°C with 100 μL of 4 μg/mL rabbit polyclonal anti-lysostaphin antibody in coating buffer (0.1 M sodium carbonate, pH 9.5). The plates were then washed with PBS-0.05% Tween 20 (PBST) 3 times, blocked for 4 hr at 37°C with 200 μL 3% BSA in PBS, and again washed 3 times. Serial dilutions of standards (recombinant lysostaphin) and samples (1:5 dilution) in dilution buffer (PBS-0.01% Tween 80 and 0.1% BSA) were added to the wells and the plates were incubated at 37°C for 1 hr, then washed with PBST 5 times. Polyclonal anti-lysostaphin antibody conjugated to horseradish peroxidase (HRP) (lightening-link HRP) at 1 μg/mL in dilution buffer (100 μL) was added and incubation was continued for 1 hr at 37°C, followed by washing 10 times with PBST and incubation with TMB for colorimetric detection. Lysostaphin was quantified by reading the absorbance at 370 nm using an Envision 2104 multilabel (Perkin Elmer).
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