Purified mScarlet-labeled NuMA C-terminal truncations. (A) Coomassie Blue-stained SDS-PAGE of the purified NuMA C-terminal truncations. The expected molecular weight (kDa) of each NuMA construct is indicated; double bands are due to the mScarlet tag. (B) Comparison of Coomassie Blue staining and mScarlet fluorescent signal for samples described in A, unboiled (U), or boiled (B) in SDS sample buffer. When the protein is not boiled, it is mostly present in a state of lower apparent molecular weight (blue arrowhead), which corresponds to an mScarlet conformation preserving its fluorescence. On the contrary, upon boiling, the most abundant band is the one with higher apparent molecular weight (red arrowhead), which corresponds to a denatured mScarlet that does not fluoresce. A minor portion of protein is resistant to denaturation, which explains the double band pattern of all mScarlet-tagged NuMA proteins. (C) Analysis of the oligomeric state of mScarlet-tagged NuMA C-terminal constructs by mass photometry. Profiles indicate the calculated molecular weight, with the associated standard deviation (σ), of the most abundant species present in each sample. The symmetric parts of the histograms around 0 kDa represent the background signal of the buffer. In the case of NuMAC-term L, the two peaks of almost equal height represent a mixed population of monomers and dimers. Source data are available for this figure: SourceData FS3.
