βIII-tubulin

Protein Overview

TUBB3 encodes a neuron-enriched β-tubulin isotype that heterodimerizes with α-tubulin to form microtubules. It is required for axon guidance, outgrowth, and maintenance during neurodevelopment, and is one of the earliest markers of neuronal lineage commitment; it also contributes to mitotic spindle formation in some non-neuronal tumor cells.

Expected Localization

Cytoplasmic, filamentous microtubule network enriched in axons and neurites, producing a fibrillar/reticular immunofluorescence pattern extending through neuronal processes and perikarya.

In dividing TUBB3-positive cells, staining also decorates the mitotic spindle.

Expected Expression Pattern

Expressed almost exclusively in neurons and neuronal progenitors (CNS and peripheral) and in testicular germ cells; largely absent from glial cells. Aberrant expression is reported in several epithelial tumors (e.g., NSCLC, ovarian cancer), associated with taxane chemoresistance.

Relevant Pathways

Microtubule polymerization/dynamics and cytoskeletal organization
Axon guidance and neurite outgrowth
Neuronal differentiation and neurogenesis
Mitotic spindle assembly in proliferating/tumor contexts
Taxane/microtubule-targeting drug resistance in cancer

HMC3 cells (microglia) were cultured in a 12-well format Cytos slide kit overnight then fixed with 4% formaldehyde for 30 minutes, permeabilized using 70% ethanol for 10 minutes. 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. βIII-tubulin demonstrated strong microtubule localization 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. SH-SY5Y, HeLa, A549, fibroblast (BJ), Jurkat + Raji (50:50 mixed well), and HepG2 cells were also cultured and processed under the same protocol in parallel as part of Cytos cross-run reproducibility testing.

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

Pass

17.3 ± 0.4 (SH-SY5Y; n=3 runs)
7.8 ± 1.7 (HeLa; n=17 runs)
6.8 ± 1.8 (A549; n=15 runs)
11.1 ± 1.5 (Fibroblast (BJ); n=3 runs)
11.4 ± 1.3 (Jurkat + Raji, 50:50 mixed well; n=3 runs)
6.1 ± 0.4 (HepG2; n=3 runs)
4.36 ± 1.43 (HMC3; n=2 runs)

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. Data shown are mean ± standard deviation across independent reproducibility runs. The result of 17.27 in the neuronal SH-SY5Y line remains the highest, with values of 6–11 across the five non-neuronal lines tested (HeLa, A549, Fibroblast, a 50:50 Jurkat+Raji well, and HepG2) — in all cases well above the acceptance threshold.

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.

HCM3	HCM3
HCM3	HCM3

Protein Overview

Clone Information

Qualitative Results

Quantitative Results

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