Phospho-ERK1 (T202) + Phospho-ERK2 (T185)

Protein Overview

ERK1/2 are terminal kinases of the RAS-RAF-MEK-ERK cascade. Dual phosphorylation within the activation loop (Thr202/Tyr204 on ERK1; Thr185/Tyr187 on ERK2) is catalyzed exclusively by MEK1/2 and is required for full catalytic activation, so this phospho-specific reagent reports the active state of ERK1/2 signaling.

Expected Localization

Inactive ERK1/2 resides predominantly in the cytoplasm; upon dual phosphorylation, active ERK1/2 rapidly translocates to the nucleus to phosphorylate transcription factors, while a pool remains active in the cytoplasm.

IF of stimulated cells shows a shift from diffuse cytoplasmic to strong nuclear signal.

Expected Expression Pattern

ERK1/2 are ubiquitously expressed; baseline phospho-signal is low in quiescent/serum-starved cells and is strongly and rapidly (5-15 min) induced by growth factor stimulation (EGF, serum, PDGF), phorbol esters (PMA), or GPCR-linked stimuli, declining over 30-60 min as phosphatases act.

Relevant Pathways

RAS/RAF/MEK/ERK (canonical MAPK) cascade
Receptor tyrosine kinase (EGFR, FGFR, insulin/IGF-1R) signal transduction
GPCR/PKC-mediated ERK activation
Cell proliferation and G0/G1 cell cycle entry
Cell differentiation and survival signaling

HeLa cells were cultured in a 12-well format Cytos slide kit overnight, treated with 100 ng/mL TNFα for 30 min, fixed with 4% formaldehyde for 30 minutes, permeabilized using 70% ethanol for 10 minutes, and then treated with Lambda Phosphatase (LPP, NEB P0753L) at 30 °C for 1 hour. After that, cells were incubated with probe made with biotinylated rabbit monoclonal anti-Phospho-ERK1 (T202) + Phospho-ERK2 (T185) antibody for 1 hour at room temperature. Negative control probe based on Biotin Rabbit Isotype Control (ABclonal AC042BC) was included to assess background signal and potential crosstalk, with no detectable crosstalk observed. The slide kit was then assembled into a flow cell and loaded onto an AVITI24™ along with the Cytos Protein Cartridge for a multiomics cell screening run. Phospho-ERK1 (T202) + Phospho-ERK2 (T185) demonstrated strong nuclear localization, digestion by LPP, and high signal-to-noise ratio, with no detectable probe crosstalk. These results support high confidence in target-specific detection and validate this antibody as a passing clone.

Signal-to-noise ratio
Acceptance criteria: > 2x background

Pass

12.6 (HeLa, TNFα 30 min, −LPP)
2.2 (HeLa, TNFα 30 min, +LPP)

Indicates the fold brightness of the target signal as measured by the Mean Intensity Above (Otsu-defined) Threshold (IAT), over the background (a channel-matched isotype control) in the same cell. The ratio of 12.60 in TNFα-stimulated HeLa cells without phosphatase treatment is over 6 times the acceptance threshold and falls to 2.17 after Lambda Phosphatase (LPP) digestion of the same TNFα-stimulated sample, confirming that the signal is phospho-specific.

Panel-level run-to-run reproducibility
Acceptance criteria: R² > 0.9

Pass

For each imaging panel, we assess reproducibility by comparing the intensity signal a given antibody target produces across independent replicate runs. Within each run, we measure the average per-well fluorescence intensity for every target in the panel. We then plot each target's intensity from one run against the matching target's intensity from another run of the same layout, and calculate the Pearson correlation coefficient (r) between those two sets of measurements. Squaring that value gives the R² (coefficient of determination), which reports what fraction of the variation in one run's signal is explained by the other run's signal.

Protein Overview

Clone Information

Qualitative Results

Quantitative Results

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