The method used was essentially as previously described [43 (link)]. T24 or HEK293 cells (30,000 cells/100 μl) were seeded in a 96-well plate in complete growth medium. After cell attachment, medium was removed and cells were serum-starved overnight in 90 μl medium without fetal bovine serum. Agonists were stored cold as 5 mM stock solutions in DMSO and diluted in HANK's buffer. DMSO control (final concentration 0.1% corresponding to the highest concentration of agonist that was used) and agonist (10 μl) were added in a 10-fold concentrated solution in Hank's medium, and cells were stimulated for 5 min. Medium was removed and cells were lysed with 1× Lysis buffer (20 μl) (PerkinElmer AlphaScreen SureFire p-ERK1/2 (Thr202/Tyr204) Assay Kit). Lysate (4 μl/well) was transferred to a 384 well ProxiPlate Plus (PerkinElmer). Acceptor Beads were diluted 1:50 in a 1:5 mixture of Activation buffer in Reaction Mix and added to the 384-well plate (5 μl/well). The plate was sealed and incubated for 2 h at room temperature. Donor beads (2 μl) diluted 1:20 in Dilution buffer were added, and the plate was incubated for another 2 h at room temperature. The plate was measured using an EnVision multilabel reader using standard AlphaScreen settings.
Alphascreen surefire p erk1 2 thr202 tyr204 assay kit
Manufactured by PerkinElmer
Sourced in United States
The AlphaScreen SureFire p-ERK1/2 (Thr202/Tyr204) Assay Kit is a laboratory tool used to detect and quantify the levels of phosphorylated ERK1/2 protein in cell-based samples. It utilizes the AlphaScreen bead-based technology to generate a luminescent signal proportional to the amount of the target analyte present in the sample.
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6 protocols using alphascreen surefire p erk1 2 thr202 tyr204 assay kit
1
Quantitative Signaling Pathway Assay
2
Quantifying ERK1/2 Activation in Cell Lines
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The method used was essentially as previously described [24]. H9C2 cells or HEK293 cells (30,000 cells/100μL) were seeded in a 96-well plate in complete growth medium. After cell attachment, the medium was removed, and cells were serum starved overnight in 90 μL of serum-free medium. For A2BAR agonist-induced ERK1/2 stimulation, cells were stimulated with agonist for 5 min. For PMA-PKC–mediated ERK1/2 stimulation, cells were pretreated with PMA for 10 min or longer. A2BAR antagonists or PKC inhibitors, GO6983, H89, or PSB603, were preincubated with cells for 20 min before addition of agonists, except that PTX (200 ng/mL) was incubated overnight. After agonist treatment, the medium was removed, and cells were lysed with 1× lysis buffer (20 μL) [AlphaScreen SureFire p-ERK1/2 (Thr202/Tyr204) Assay Kit; PerkinElmer]. Lysate (4 μL/well) was transferred to a 384-well ProxiPlate Plus (PerkinElmer). Acceptor beads were diluted 1:50 in a 1:5 mixture of activation buffer in reaction mix and added to the 384-well plate (5 μL/well). The plate was sealed and incubated for 2 h at room temperature. Donor beads (2 μL) diluted 1:20 in dilution buffer were added, and the plate was incubated for another 2 h at room temperature. The plate was measured using an EnVision multilabel reader using standard AlphaScreen settings.
3
Quantifying Cellular pERK1/2 Activation
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To measure pERK1/2 we utilised the high throughput AlphaScreen SureFire pERK1/2 (Thr202/Tyr204) Assay Kit (PerkinElmer)42 (link). Detection of pERK1/2 is enabled by immuno-sandwich capture of endogenous pERK1/2 in cell lysates. Antibody-coated AlphaScreen beads generate a highly amplified signal when near due to binding of pERK1/2. Cells were seeded at 50,000 cells/well in a 96 well tissue culture plate and incubated for 24 hours at 37 °C. Cells were then incubated in serum free media overnight. Test compounds were added to the cells and incubated for 10 min at room temperature before cells were lysed and assayed for pERK1/2 using the AlphaScreen-based detection kit according to the manufacturer’s protocol (PerkinElmer).
4
Measurement of pERK1/2 in HUVEC
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HUVEC were seeded onto non-coated 96-well plates (15,000 cells/well) and cultured for 48 h. Cells were serum starved for 6 h and treated as described in the results section. Phospho-ERK 1/2 (pERK1/2) was measured using the AlphaScreen SureFire p-ERK 1/2 (Thr202/Tyr204) Assay Kit (PerkinElmer, USA), according to the manufacturer’s specifications. Fluorescence was measured using the EnVision multilabel plate reader (PerkinElmer). Data were normalized to the positive control (PDBu, 1 µM).
5
Monitoring ERK1/2 and Akt Activation
6
ERK1/2 Activation Assay Protocol
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The method used was essentially as previously described.23 (link) H9C2 cells or HEK293 cells (30,000 cells/100 μL) were seeded in a 96-well plate in complete growth medium. After cell attachment, the medium was removed, and cells were serum starved overnight in 90 μL of serum-free medium. For A2BAR agonist-induced ERK1/2 stimulation, cells were stimulated with agonist for 5 min. For PMA-PKC–mediated ERK1/2 stimulation, cells were pretreated with PMA for 10 min or longer. A2BAR antagonists or PKC inhibitors, GO6983, H89, or PSB603, were preincubated with cells for 20 min before addition of agonists, except that PTX (200 ng/mL) was incubated overnight. After agonist treatment, the medium was removed, and cells were lysed with 1× lysis buffer (20 μL, AlphaScreen SureFire p-ERK1/2 (Thr202/Tyr204) Assay Kit; PerkinElmer). Lysate (4 μL/well) was transferred to a 384-well ProxiPlate Plus (PerkinElmer). Acceptor beads were diluted 1:50 in a 1:5 mixture of activation buffer in reaction mix and added to the 384-well plate (5 μL/well). The plate was sealed and incubated for 2 h at room temperature. Donor beads (2 μL) diluted 1:20 in dilution buffer were added, and the plate was incubated for another 2 h at room temperature. The plate was measured using an EnVision multilabel reader using standard AlphaScreen settings.
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