384 well proxiplate

Manufactured by PerkinElmer

Sourced in United States

The 384-well ProxiPlates are a high-density microplate designed for a variety of assay applications. They feature 384 individual wells in a standard microplate footprint, enabling efficient use of sample and reagent volumes. The ProxiPlates are made from high-quality, durable materials to provide consistent and reliable performance in your laboratory.

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Lab products found in correlation

9 protocols using 384 well proxiplate

Quantifying Glucagon-Like Peptide-1 Receptor Signaling

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For tissue culture Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 medium, hygromycin-B, and Fluo-4 acetoxymethyl ester were used (Invitrogen Carlsbad, CA). AlphaScreen reagents, ¹²⁵I-Ex(9–39), ¹²⁵I-hGLP-1 and 384-well ProxiPlates were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). hGLP-1, pGLP-1, eGLP-1 and Ex-4 were purchased from GL Biochem (Shanghai) Ltd. (Shanghai, China). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) or BDH Merck (Melbourne, VIC, Australia) and were of an analytical grade. The parental INS-1 (832/13) cell line was kindly provided by Chris Newgard40 (link).

Tsend-Ayush E., He C., Myers M.A., Andrikopoulos S., Wong N., Sexton P.M., Wootten D., Forbes B.E, & Grutzner F. (2016). Monotreme glucagon-like peptide-1 in venom and gut: one gene – two very different functions. Scientific Reports, 6, 37744.

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Fluorescent Exendin-4 Assay Protocol

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DMEM, PBS, Hanks' buffered salt solution (HBSS), Lipofectamine^TM reagent, PLUS^TM reagent, NuPAGE® 4–12% BisTris protein gels and buffers, DynaBeads® protein G, chemiluminescent substrate, and monoclonal mouse anti-V5 Ab (R960-25) were purchased from Life Technologies, Inc. Polyclonal rabbit anti-FL Ab (ab19491) was purchased from Abcam (Cambridge, MA). The QuikChange® Lightning site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA). AzF was purchased from Chem Impex International (Wood Dale, IL). Exendin-4 was purchased from American Peptide (Sunnyvale, CA). FL-labeled exendin-4 analogues were purchased from Anaspec (Fremont, CA) and NeoScientific (Cambridge, MA). AlphaScreen^TM reagents and 384-well ProxiPlates were purchased from PerkinElmer Life Sciences. SigmaFast^TMo-phenylenediamine dihydrochloride tablets, n-dodecyl β-d-maltoside (DM), and monoclonal mouse anti-c-Myc Ab (M4439, clone 9E10) were purchased from Sigma. PVDF membranes were purchased from EMD Millipore (Billerica, MA). HyBlot CL autoradiography film was purchased from Denville Scientific (Holliston, MA). FBS was purchased from Gemini Bioproducts (West Sacramento, CA). All other reagents were obtained from Thermo Fisher Scientific (Waltham, MA) and were of analytical grade.

Koole C., Reynolds C.A., Mobarec J.C., Hick C., Sexton P.M, & Sakmar T.P. (2017). Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R). The Journal of Biological Chemistry, 292(17), 7131-7144.

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Quantitative p53 Protein Analysis

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The levels of p53 protein were quantitatively analysed using the Alphascreen SureFire Total p53 Assay Kit (TGR Biosciences/Perkin Elmer #TGRT53). Briefly, A549 cells (40,000 cells/well) were reverse transfected with 40 nM siRNA (final reaction volume 0.5 mL) in Nunc 24-well cell culture plates for 72 hours, refreshing media at 24 hours’ post transfection. Just prior to harvest, to normalise p53 signal, plates were imaged on the IncuCyte S3 Live-Cell Analysis Imaging System (Essen Biosciences) using the scan-on-demand feature (2015A software release), calculating the Phase Object Confluence (Percent) for each well. Media was then carefully removed, and the cell monolayers lysed with 30 μL 1x Alphascreen Lysis Buffer for 2-3 minutes on an orbital rocker, prior to transfer of lysates to −20°C. Lysates (4 μL/well in Perkin Elmer 384 well proxiplates, #6008280) were then assayed in as per the manufacturers’ instructions. Plates were then read using the Perkin Elmer EnSpire Multimode Plate Reader instrument using the instrument pre-defined Alphascreen settings and the data normalised to the cell confluence readings, then normalised to the non-targeting siRNA treated wells.

Hannan K.M., Soo P., Wong M.S., Lee J.K., Hein N., Poh P., Wysoke K.D., Williams T.D., Montellese C., Smith L.K., Al-Obaidi S.J., Núñez-Villacís L., Pavy M., He J.S., Parsons K.M., Loring K.E., Morrison T., Diesch J., Burgio G., Ferreira R., Feng Z.P., Gould C.M., Madhamshettiwar P.B., Flygare J., Gonda T.J., Simpson K.J., Kutay U., Pearson R.B., Engel C., Watkins N.J., Hannan R.D, & George A.J. (2022). Nuclear stabilization of p53 requires a functional nucleolar surveillance pathway. Cell Reports, 41(5), 111571.

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IL-17A/IL-17RA Binding Inhibition Assay

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The ability of compounds to inhibit the binding of IL-17A to IL-17RA was followed with an AlphaLISA assay. 0.1 μl of compounds in 100% DMSO were serial diluted using Echo Acoustic Liquid Handler (LabCyte Sunnydale, CA) into 384 well proxiplates (Perkin Elmer). A 5 ul addition of IL-17RA-flag was added to the plate, followed by the addition of 5 ul Avi-tagged IL-17A-His to provide a final concentration of 2 nM of IL-17RA and IL-17A, respectively. The assay was carried out in 20mM HEPES, 150mM NaCl, 0.05% Tween-20, 0.1%Human Serum Albumin, pH 7and 1% DMSO. The assay mixture was allowed to incubate at room temperature for 2hours. After the 2 hour incubation 10uL of streptavidin donor beads (20ug/ml final) and Anti-FLAG acceptor beads (20 ug/ml final) were added to the plate. The plate was allowed to incubate for one hour at room temp, then read on the Envision (Perkin Elmer). The relative IC50 values were determined using a four parameter fit as shown in Eq 1. In this equation, bottom and top are defined as the plateaus of the curve, and H is the Hill Slope.
No inhibitory effect was observed from addition of 0.1% DMSO only.

Ting J.P., Tung F., Antonysamy S., Wasserman S., Jones S.B., Zhang F.F., Espada A., Broughton H., Chalmers M.J., Woodman M.E., Bina H.A., Dodge J.A., Benach J., Zhang A., Groshong C., Manglicmot D., Russell M, & Afshar S. (2018). Utilization of peptide phage display to investigate hotspots on IL-17A and what it means for drug discovery. PLoS ONE, 13(1), e0190850.

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GLP-1 Receptor Signaling Assay

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Dulbecco’s modified Eagle’s medium (DMEM), hygromycin-B and Fluo-4 acetoxymethyl (AM) ester were purchased from Invitrogen (Carlsbad, CA, USA). Foetal bovine serum (FBS) was purchased from Thermo Fisher Scientific (Melbourne, VIC, Australia). The QuikChange™ site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA, USA). AlphaScreen™ reagents, Bolton-Hunter reagent [¹²⁵I] and 384-well ProxiPlates were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA, USA). SureFire™ ERK1/2 reagents were generously supplied by TGR Biosciences (Adelaide, SA, Australia). SigmaFast o-phenylenediamine dihydrochloride (OPD) tablets and antibodies were purchased from Sigma–Aldrich (St. Louis, MO, USA). GLP-1 peptides were purchased from Mimotopes (Clayton, VIC, Australia). All other reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA) or BDH Merck (Melbourne, VIC, Australia) and were of an analytical grade.

Wootten D., Reynolds C.A., Smith K.J., Mobarec J.C., Furness S.G., Miller L.J., Christopoulos A, & Sexton P.M. (2016). Key interactions by conserved polar amino acids located at the transmembrane helical boundaries in Class B GPCRs modulate activation, effector specificity and biased signalling in the glucagon-like peptide-1 receptor. Biochemical Pharmacology, 118, 68-87.

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Synthesis and Characterization of Novel Compounds

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Aripiprazole was synthesized in house as previously described and shown to be >98% pure.^{43 (link)} Ropinirole was purchased from BetaPharma Co. Ltd. (Wujiang, China) and was >98% pure as described by the supplier. All novel compounds were synthesized as described in the Supporting Information. pcDNA3l-His-CAMYEL was purchased from ATCC. Dulbecco’s modified Eagle’s medium (DMEM), hygromycin B, and FlpIn CHO cells were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from ThermoTrace (Melbourne, Australia). [³H]Spiperone, [³H]raclopride, Alpha-Screen reagents, Ultima gold scintillation cocktail, 384-well optiplates, and 384-well proxiplates were purchased from PerkinElmer (Boston, MA). All of the other reagents were purchased from Sigma-Aldrich (Castle Hill, Australia).

Herenbrink C.K., Verma R., Lim H.D., Kopinathan A., Keen A., Shonberg J., Draper-Joyce C.J., Scammells P.J., Christopoulos A., Javitch J.A., Capuano B., Shi L, & Lane J.R. (2019). Molecular Determinants of the Intrinsic Efficacy of the Antipsychotic Aripiprazole. ACS chemical biology, 14(8), 1780-1792.

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Synthesis and Characterization of SB269652 Derivatives

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SB269652 and all derivatives were synthesised as previously described and were shown to be >95% pure [8 (link),9 (link)]. Dopamine was purchased from Sigma-Aldrich (Castle Hill, NSW, Australia) and was >98% pure as indicated by the supplier. Dulbecco’s modified Eagle’s medium (DMEM), hygromycin B, and FlpIn CHO cells were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from ThermoTrace (Melbourne, VIC, Australia). [³H]Spiperone, AlphaScreen reagents, Ultima gold scintillation cocktail, and 384-well proxiplates were purchased from PerkinElmer (Boston, MA). All the other reagents were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia).

Draper-Joyce C.J., Michino M., Verma R.K., Herenbrink C.K., Shonberg J., Kopinathan A., Scammells P.J., Capuano B., Thal D.M., Javitch J.A., Christopoulos A., Shi L, & Lane J.R. (2018). The structural determinants of the bitopic binding mode of a negative allosteric modulator of the dopamine D2 receptor. Biochemical pharmacology, 148, 315-328.

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Fluorescent Ligand Binding Assay for β2-Adrenergic Receptor

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Solubilised Lumi4-Tb labelled β2AR was incubated with 10μM BODIPY™ FL L-Cystine dye (Molecular Probes, U.S) with or without 200nM F-propranolol or 100μM cyanopindolol, for 15 mins on ice in 20mM HEPES, 150mM NaCl, 5% glycerol, 0.5% BSA, pH8. For DDM samples 0.1% DDM was used. 20μL samples were added to each well of a 96-well PCR plate and incubated for 30 min over a temperature gradient of 20-78°C across the plate using alpha cycler 2 PCR machine (PCRmax, U.K). Samples were transferred to a 384-well proxiplate (PerkinElmer, U.S). TR-FRET between BODIPY™ FL L-Cystine dye and Lumi4-Tb was read by exciting at 337nm and reading emission at 620nm and 520nm using Pherstar FSX and 337 520/620 module (BMG Labtech). F-propranolol and fluorescent XAC (F-XAC) (CellAura, UK) binding was measured using HTRF 337 665/620 module as above.

Harwood C.R., Sykes D.A., Hoare B.L., Heydenreich F.M., Uddin R., Poyner D.R., Briddon S.J, & Veprintsev D.B. (2021). Functional solubilization of the β2-adrenoceptor using diisobutylene maleic acid. iScience, 24(12), 103362.

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Fluorescent Ligand Binding Assay for β2AR

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Harwood C.R., Sykes D.A., Hoare B.L., Heydenreich F.M., Uddin R., Poyner D.R., Briddon S.J., & Veprintsev D.B. (2020). Functional solubilisation of the β₂-adrenoceptor (β₂AR) using Diisobutylene maleic acid (DIBMA).

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