Alphascreen surefire p erk assay kit

A₃-YFP or A₃ W243F-YFP CRE-SPAP cells were grown to confluency in clear 96-well plates. Normal growth medium was replaced with serum-free medium for at least 4 h before analysis. Levels of ERK1/2 phosphorylation were measured using the AlphaScreen SureFire p-ERK assay kit (PerkinElmer). Briefly, cells were stimulated with the required concentration of NECA for 5 min in fresh serum-free medium. Medium was removed from each well and replaced with 40 μL SureFire lysis buffer. After shaking for 5 min, a 1:80:20:120 v v⁻¹ dilution of AlphaScreen beads : lysate : activation buffer : reaction buffer in a 5.5 μL total volume was transferred to a white opaque 384-well proxiplate in diminished light. After 2 h of incubation in the dark at room temperature, the fluorescence signal was measured with an EnVision plate reader (PerkinElmer) using standard AlphaScreen settings.

G418, Lipofectamine and Optimem were obtained from Invitrogen (Paisley, UK). FCS was obtained from PAA Laboratories (Wokingham, UK), L-glutamine from Lonza (Basel, Switzerland) and Fugene HD from Promega (Southampton, UK). Pertussis toxin (PTx) was purchased from Merck Chemicals (Nottingham, UK). NECA [5-(N-ethylcarboxamido)adenosine] and HEMADO [2-(1-hexynyl)-N-methyladenosine] were obtained from Tocris Bioscience (Bristol, UK). The AlphaScreen SureFire p-ERK assay kit was obtained from PerkinElmer (Waltham, MA, USA). [³H]-adenine and [¹⁴C]-cAMP were from Perkin-Elmer (Buckinghamshire, UK). CA200645 was purchased from Cellaura Technologies Ltd (Nottingham, UK). All other chemicals and reagents were purchased from Sigma-Aldrich (Gillingham, UK).

MAP kinase activation was monitored using the bead proximity-based AlphaScreen assay to detect phosphorylated ERK (SureFire p-ERK, PerkinElmer, Boston, MA). Virally transduced HEK-293/EBNA cells were seeded in 96 well plates with serum-free growth medium at 50,000 cells/well, followed by incubation for six hours at 37°C in 7% CO₂. Cells were then stimulated with vehicle or agonist for 5 min at 37°C in 7% CO₂. Agonist response was terminated following rapid removal of the medium by adding 50 μL per well of SureFire lysis buffer, followed by incubation of cells for 10 min at room temperature. Plates were stored at −80 C prior to analysis for p-ERK levels. Processing of cells for phospho-ERK detection was performed using an AlphaScreen SureFire p-ERK assay kit (PerkinElmer, Boston, MA) according to specifications from the manufacturer. Briefly, 10 μL of lysate were transferred to 384 well plates (OptiPlate) and combined with 17 μL of SureFire buffer containing AlphaScreen beads. Plates were incubated for 2 h at room temperature and the fluorescence signal was recorded using a Fusion plate reader (PerkinElmer), adjusted to standard AlphaScreen settings.