To assess if the source of the observed interblastomeric variation in β-tubulin AUC was attributable to biological variation or confounding technical variation, we first sought to deconvolve sources of variation arising from preparatory steps (e.g., cell lysis) from those arising from analytical steps (e.g., PAGE, photoblotting, and immunoprobing). Variation in measured protein expression levels originates from biological sources, technical sources, or a combination. In the analytical steps, the sources of variation are predominantly technical. Consequently, to estimate the technical variation threshold for these downstream analytical steps, we used a well-characterized method using purified proteins (20, 21) (bead-based delivery of protein ladders is also possible (59)) that allows us to establish a technical variation threshold by quantifying immunoblots of microwells uniformly loaded with purified protein. Given that endogenous loading control protein targets (i) show significant cell-to-cell variation (60) or (ii) form dimers that are difficult to solubilize (61), we utilize purified protein to estimate technical variation as we have previously reported (62). Briefly, we partitioned a solution of purified bovine serum albumin (BSA) (1 μM in PBS) into the microwells by incubating PA gels in BSA solution for 30 min. We then performed the immunoblotting assay and quantified the AUC for each BSA protein band. We calculated the coefficient of variation in BSA AUC (CVAUC % = AUC SD/mean AUC × 100) and computed a technical variation threshold defined as >3 × SD of the mean CVAUC (CVthreshold = mean CVAUC + 3 SD = 7.4%, where mean CVAUC = 4.69% and SD = 0.92%; fig. S6) (62) .

In single-cell resolution assays, sources of variation in cell preparatory steps become nuanced. Cell lysis is the dominant preparatory step for the single-cell immunoblot, and lysis behavior is influenced by the biochemical (e.g., fluidity or membrane composition (63)) and biomechanical (e.g., changes in viscoelasticity (64)) properties of each cell, which can vary extensively in embryos (64). Consequently, given the established link between cellular properties and cellular lysis behavior, we primarily attribute variation in cell lysis behavior to biological sources. As such, including cell lysis variability in estimates of the technical variation threshold is likely to be overly conservative. Nevertheless, a more conservative technical variation threshold, at 11%, can be estimated when the impact of variable lysis behavior is included in protein expression variation of an enhanced green fluorescent protein–expressing cell line (62). When comparing the intraembryonic variation in β-tubulin expression to the conservative average coefficient of variation of 11% (includes variation from cell lysis, which is arguably biological in origin), we observe a majority of embryos having variation larger than the threshold value (two of three embryos, n = 4 blastomeres per embryo; fig. S1D).

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