Alphascreensurefire p erk1 2 kit

Manufactured by PerkinElmer

The AlphaScreenSureFire p-ERK1/2 kit is a bead-based detection assay that measures the phosphorylation of ERK1/2 proteins. It provides a sensitive and quantitative method for analyzing the activation of the ERK signaling pathway in cell-based samples.

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Lab products found in correlation

Fusion α plate reader, by PerkinElmer (1 mentions) Proxiplate, by PerkinElmer (1 mentions) Bright glo luciferase reagent, by Promega (1 mentions)

2 protocols using alphascreensurefire p erk1 2 kit

Measuring DOR-Mediated ERK1/2 Phosphorylation

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Cells were seeded into transparent
96-well plates at 50 000 per well, allowed to adhere for 6
h, and then serum starved overnight. Previous studies have shown that
maximal stimulation of DOR by SNC-80 is achieved after 5 min.^{13 (link)} Thus, cells were stimulated with ligands for
5 min at 37 °C in 5% CO₂. For interaction studies
with BMS-986187, increasing concentrations of SNC-80 and allosteric
ligand were added simultaneously. The reaction was terminated by removal
of media and ligands, and the samples were processed using the AlphaScreen
SureFire p-ERK1/2 kit (PerkinElmer) as per the manufacturer’s
instructions. The fluorescence signal was measured using a Fusion-α
plate reader (PerkinElmer). Data were normalized to the maximal response
elicited by 10% v/v FBS at the same time point.

Shang Y., Yeatman H.R., Provasi D., Alt A., Christopoulos A., Canals M, & Filizola M. (2016). Proposed Mode of Binding and Action of Positive Allosteric Modulators at Opioid Receptors. ACS Chemical Biology, 11(5), 1220-1229.

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Measuring ERK1/2 phosphorylation and cAMP response

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Changes in ERK1/2 phosphorylation (pERK) were detected using the AlphaScreen SureFire pERK1/2 Kit (PerkinElmer). Cells were treated with carvedilol or phorbol-12,13-dibutyrate (PDBu) diluted in serum-free medium for 30 min at 37°C. Medium was aspirated, cells were lysed in 100 ml lysis buffer, and cell lysates (4 ml) were transferred to 384-well ProxiPlates (PerkinElmer) for detection. Data are expressed relative to the effect of serum-free medium.
cAMP Response Element reporter assay MDA-MB-231 cells stably transfected with pCRE-Luc were seeded in a 96 well plate overnight. 24 hours later the cells were serum starved overnight. Following serum starving cells were treated with isoprenaline and/or carvedilol (all 1 µM, in 0.1% DMSO in serum-free medium) for 6 hours. When used as an antagonist, cells were treated with carvedilol for 30 minutes prior to the addition of isoprenaline. After incubation, the media was removed and luminescence was quantified using the Bright Glo luciferase reagent (Promega, USA) and an EnVision plate reader.

Gillis R.D., Botteri E., Chang A., Ziegler A.I., Chung N.C., Pon C.K., Shackleford D.M., Andreassen B.K., Halls M.L., Baker J.G, & Sloan E.K. (2021). Carvedilol blocks neural regulation of breast cancer progression in vivo and is associated with reduced breast cancer mortality in patients. European journal of cancer (Oxford, England : 1990), 147.

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