Alexa fluor 488 alexa488

Capped RP51A pre-mRNA for in vitro splicing assays were transcribed in the presence of [α-³²P]UTP and purified as previously described (Crawford et al., 2008 (link)). Dye-labeled RP51A pre-mRNAs used in single molecule experiments were prepared by splinted ligation of a trace [³²P]-labeled, capped RP51A transcript to a biotinylated 2'-O-methyl oligonoucleotide derivatized with a single Alexa Fluor 488 (Alexa488, ThermoFisher Scientific; Waltham, MA) or Alexa Fluor 647 (Alexa647, ThermoFisher Scientific; Waltham, MA) fluorophore as previously described (Crawford et al., 2008 (link)).

To increase contrast during fluorescence imaging, Cy5 (free or RNA-labelled), Alexa Fluor 488 (Alexa 488; Thermo Fisher Scientific) or mCitrine (YFP), were included at final concentrations of 1 μM, 0.6 μM and 2 μM, respectively. Images were captured at room temperature using a Leica TCS-SP5 confocal fluorescence microscope and an HCX PL APO CS 63x (NA 1.40) oil immersion objective. Samples were illuminated with 488 nm (Alexa 488), 514 nm (YFP), or 633 nm (Cy5) lasers, with power and gain settings adjusted to give mean fluorescence intensity values of protein condensates of approximately 50–70% of the maximum 16-bit depth (∼32,000–46,000 a.u.). Images were typically captured with settings of 256 × 256 pixels at ∼ 98 × 98 × 98 nm (XYZ) resolution, 1400 Hz scan speed and a line average of 2.
Phase separation was initiated by mixing protein solutions with a buffer of lower ionic strength, as described above, and were left for 10 min to promote droplet growth. Phase separated solutions were then transferred to a 0.22 mm thick siliconized glass coverslip (Hampton Research), before sealing with 0.12 mm imaging spacers (Sigma) and a second siliconized glass coverslip. Samples were then left to equilibrate for approximately 50 min prior to imaging.

Capped [³²P]-labeled RP51A pre-mRNAs for in vitro splicing and trace [³²P]-labeled RP51A substrates for single molecule assays were made by in vitro transcription with T7 RNA polymerase in the presence of [α-³²P] UTP and G(5’)ppp(5’)G RNA cap analog (NEB). Trace [³²P]-labeled RP51A was fluorescently labeled by splinted ligation with a biotinylated 2’-O-methyl oligonucleotide derivatized with a single Alexa Fluor 488 (Alexa488, ThermoFisher Scientific) or Cy5 (GE Life Sciences) fluorophore as previously described.