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The limit of detection was defined as 3 standard deviations for the field blank samples as described in 4

The 1.53 M concentrations of SERF2 that allow phase separation in the presence of rG4s are below the SERF2 concentrations present in human cells which range from ~7 M in U2OS cells to ~23 M in MCF7 breast cancer cells 61

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Each freeze-thaw event introduces micro-crystallisation stress on the protein structure, and repeated cycles accumulate damage that accelerates degradation once the peptide is reconstituted