Black low volume 384 well plates, by Greiner - Product details

1

TR-FRET Assay for REV-ERB Ligand Binding

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The TR-FRET assay was performed in black low-volume 384-well plates (Greiner). Each well contained 4 nM 6xHistag-REV-ERBα LBD (human; residues 281–614) or 6xHistag-REV-ERBβ LBD (human; residues 212–579) protein expressed in and purified from Escherichia coli using nickel affinity and size exclusion chromatography; 1 nM LanthaScreen Elite Tb-anti-HIS Antibody; and 400 nM FITC-labeled peptide derived from the SMRT corepressor containing a N-terminal FITC label with a six-carbon linker (Ahx) and an amidated C terminus for stability in TR-FRET buffer (20 mm potassium phosphate, pH 7.4, 50 mm potassium chloride, 1 mm dithiothreitol, and 0.005% Tween-20). Ligand stocks were prepared via serial dilution in DMSO, added to wells in triplicate, and plates were incubated at 4°C for 2 h and read using BioTek Synergy Neo multimode plate reader. The Tb donor was excited at 340 nm; its fluorescence emission was monitored at 495 nm, and the acceptor FITC emission was measured at 520 nm; and the TR-FRET ratio was calculated as the signal at 520 nm divided by the signal at 495 nm. Data were plotted using GraphPad Prism as TR-FRET ratio versus ligand concentration and fit to sigmoidal dose response curve equation.

Amir M., Chaudhari S., Wang R., Campbell S., Mosure S.A., Chopp L.B., Lu Q., Shang J., Pelletier O.B., He Y., Doebelin C., Cameron M.D., Kojetin D.J., Kamenecka T.M, & Solt L.A. (2018). REV-ERBα Regulates TH17 Cell Development and Autoimmunity. Cell reports, 25(13), 3733-3749.e8.

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TR-FRET Assay for REV-ERB Ligand Binding

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The TR-FRET assay was performed in black low-volume 384-well plates (Greiner). Each well contained 4 nM 6xHistag-REV-ERBα LBD (human; residues 281–614) or 6xHistag-REV-ERBβ LBD (human; residues 212–579) protein expressed in and purified from Escherichia coli using nickel affinity and size exclusion chromatography; 1 nM LanthaScreen Elite Tb-anti-HIS Antibody; and 400 nM FITC-labeled peptide derived from the SMRT corepressor containing a N-terminal FITC label with a six-carbon linker (Ahx) and an amidated C terminus for stability in TR-FRET buffer (20 mm potassium phosphate, pH 7.4, 50 mm potassium chloride, 1 mm dithiothreitol, and 0.005% Tween-20). Ligand stocks were prepared via serial dilution in DMSO, added to wells in triplicate, and plates were incubated at 4°C for 2 h and read using BioTek Synergy Neo multimode plate reader. The Tb donor was excited at 340 nm; its fluorescence emission was monitored at 495 nm, and the acceptor FITC emission was measured at 520 nm; and the TR-FRET ratio was calculated as the signal at 520 nm divided by the signal at 495 nm. Data were plotted using GraphPad Prism as TR-FRET ratio versus ligand concentration and fit to sigmoidal dose response curve equation.

Amir M., Chaudhari S., Wang R., Campbell S., Mosure S.A., Chopp L.B., Lu Q., Shang J., Pelletier O.B., He Y., Doebelin C., Cameron M.D., Kojetin D.J., Kamenecka T.M, & Solt L.A. (2018). REV-ERBα Regulates TH17 Cell Development and Autoimmunity. Cell reports, 25(13), 3733-3749.e8.

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3

PPAR-γ ligand binding assay

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Fluorescence polarization assays were performed in black low-volume 384-well plates (Greiner) using a buffer containing 20 mM potassium phosphate (pH 7.4), 50 mM potassium chloride, 0.5 mM EDTA, 5 mM TCEP, and 0.01% Tween-20. Each well contained 100 nM FITC-labeled TRAP220 coactivator peptide (Lifetein), a serial dilution of PPARγ LBD (1.5 nM–90 µM), with a fixed concentration of vehicle control (1% DMSO) or ligand equal to the highest protein concentration (90 µM Pio or PioOH) in triplicate. Plates were incubated 2 hrs at 4°C and read using BioTek Synergy Neo multimode plate reader. Data were plotted in GraphPad Prism and fit to a sigmoidal dose response equation. Peptide affinity and polarization window from n=2 individual experiments was analyzed by unpaired t-test.

Mosure S.A., Shang J., Eberhardt J., Brust R., Zheng J., Griffin P.R., Forli S, & Kojetin D.J. (2019). Structural basis of altered potency and efficacy displayed by a major in vivo metabolite of the anti-diabetic PPARγ drug pioglitazone. Journal of medicinal chemistry, 62(4), 2008-2023.

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Monitoring RhoA Nucleotide Exchange

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RhoA was incubated with alkaline phosphatase to remove bound nucleotide and then purified by gel filtration under the same conditions as above. The nucleotide-free RhoA was incubated with MANT (N-methylanthraniloyl)-GDP (Life Technologies) for 15 min. The protein was then passed through a PD-10 column pre-equilibrated in GF buffer to remove excess MANT-GDP. Guanine-nucleotide-exchange assays were conducted in 20 μl aliquots in black 384-well low-volume plates (Greiner). The progress of the nucleotide exchange was monitored by fluorescence on a PheraStar plate reader (BMG Labtech) with excitation at 360 nm and detection at 440 nm. Exchange reactions were measured in GF buffer containing 10 mM MgCl₂, with 0.5 μM RhoA–MANT-GDP and 50 μM unlabelled GMP-PNP, and in the presence or absence of varying concentrations of DH or DH–PH domain protein.

Abdul Azeez K.R., Knapp S., Fernandes J.M., Klussmann E, & Elkins J.M. (2014). The crystal structure of the RhoA–AKAP-Lbc DH–PH domain complex. Biochemical Journal, 464(Pt 2), 231-239.

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5

PPARγ Competitive Binding Assay

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LanthaScreen TR-FRET PPARγ competitive binding assay
(Cat:PV4893) was performed according to the manufacturer’s protocol.
A mixture of 5 nM PPARG ligand binding domain (GST-PPARγ-LBD,
Cat:PV4545) or PPARα ligand binding domain (GST-PPARα-LBD,
Cat:PV4691), 5 μM Tb-GST-antibody (Cat:PV3550), 5 nM Fluormone
Pan-PPAR Green (Cat:PV4896), and serial dilutions of compound beginning at
10 μM downwards was added to wells of black 384-well low-volume
plates (Greiner) to a total volume of 18 μl. All dilutions were made
in TR-FRET PPAR assay buffer. DMSO at 2% final concentration was used as a
no-ligand control. Experiments were performed in triplicate and incubated
for 2h in the dark before analysis in Perkin Elmer ViewLux ultra HTS
microplate reader. The FRET signal was measured by excitation at 340 nm and
emission at 520 nm for fluorescein and 490 nm for terbium. The fold change
over DMSO was calculated by 520 nm/490 nm ratio. Graphs were plotted in
GraphPad Prism as fold change of FRET signal for each compound over
DMSO-only control. IC50s were determined from GraphPad Prism software.

Zheng J., Corzo C., Chang M.R., Shang J., Lam V.Q., Brust R., Blayo A.L., Bruning J.B., Kamenecka T.M., Kojetin D.J, & Griffin P.R. (2018). Chemical crosslinking mass spectrometry reveals the conformational landscape of the activation helix of PPARγ; a model for ligand-dependent antagonism. Structure (London, England : 1993), 26(11), 1431-1439.e6.

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Black low volume 384 well plates

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5 protocols using black low volume 384 well plates

TR-FRET Assay for REV-ERB Ligand Binding

TR-FRET Assay for REV-ERB Ligand Binding

PPAR-γ ligand binding assay

Monitoring RhoA Nucleotide Exchange

PPARγ Competitive Binding Assay

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