Microscopy images were collected on an inverted widefield fluorescence Nikon Ti-E microscope equipped with a Hamamatsu Flash4.0 camera using PlanApo 20x or 40x air objectives. Samples were imaged in a Greiner Bio-One 384-well glass bottom plate PEGylated using 20 mg/mL PEG-Silane (Laysan Bio, MPEG-SIL-5000) and passivated with 100 mg/mL BSA as described51 (link). Prior to addition of samples, the wells were washed 3x with 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT. Dcp1/Dcp2 constructs assayed for phase separation by microscopy were prepared by initiating removal of the N-terminal MBP solubility tag with 1:40 molar equivalent TEV:Dcp1/Dcp2. Dcp1/Dcp2/Edc3 droplets were prepared by incubating Dcp1/Dcp2 and Edc3 prior to removal of the N-terminal MBP tag from Dcp1/Dcp2ext. Imaging was performed after 30 minutes to ensure TEV cleavage and droplet. Image analysis was performed using ImageJ52 (link). For localization and enrichment of Dcp1/Dcp2ext, Edc3, or RNA in droplets, 1%–5% protein concentration was fluorescently labelled. Enrichment was estimated from the average ratio of intensity in at least twenty droplets (Idroplet) to average intensity in surrounding solution (Idilute).
Mpeg sil 5000
Manufactured by Laysan Bio
The MPEG-SIL-5000 is a high-performance liquid chromatography (HPLC) system designed for the separation and analysis of a wide range of chemical compounds. It features a modular design, allowing for customization to meet specific research or analytical needs.
Automatically generated - may contain errors
4 protocols using mpeg sil 5000
1
Microscopy Imaging of Dcp1/Dcp2 Phase Separation
2
Microscopy Imaging of Dcp1/Dcp2 Phase Separation
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Microscopy images were collected on an inverted widefield fluorescence Nikon Ti-E microscope equipped with a Hamamatsu Flash4.0 camera using PlanApo 20x or 40x air objectives. Samples were imaged in a Greiner Bio-One 384-well glass bottom plate PEGylated using 20 mg/mL PEG-Silane (Laysan Bio, MPEG-SIL-5000) and passivated with 100 mg/mL BSA as described51 (link). Prior to addition of samples, the wells were washed 3x with 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT. Dcp1/Dcp2 constructs assayed for phase separation by microscopy were prepared by initiating removal of the N-terminal MBP solubility tag with 1:40 molar equivalent TEV:Dcp1/Dcp2. Dcp1/Dcp2/Edc3 droplets were prepared by incubating Dcp1/Dcp2 and Edc3 prior to removal of the N-terminal MBP tag from Dcp1/Dcp2ext. Imaging was performed after 30 minutes to ensure TEV cleavage and droplet. Image analysis was performed using ImageJ52 (link). For localization and enrichment of Dcp1/Dcp2ext, Edc3, or RNA in droplets, 1%–5% protein concentration was fluorescently labelled. Enrichment was estimated from the average ratio of intensity in at least twenty droplets (Idroplet) to average intensity in surrounding solution (Idilute).
3
Functionalization of Glass Coverslips
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Coverslips (Thorlabs, CG15KH) were placed into a glass jar, bathed in acetone for 30 min, incubated in ethanol for 15 min, and washed in MiliQ water three times. Subsequently, the coverslips were sonicated in 2% Hellmanex or 2% Micro90 for 2 h at room temperature in a water bath sonicator, followed by series of washes in MiliQ water. Coverslips were then transferred into a 0.1 M KOH bath, incubated for 30 min, washed in MiliQ water and dried with clean nitrogen gas. For functionalization, coverslips were submerged in a glass jar containing mPEG-Silane (Laysan Bio Inc, MPEG-SIL-5000) or Biotin-mPEG-Silane (Laysan Bio Inc, Biotin-PEG-SIL-5K) solution, and protected from light for 18 h. The next day, coverslips were washed with clean ethanol and MiliQ water. Finally, coverslips were dried once again with clean nitrogen gas and stored at 4 °C for up to 2 weeks.
4
Microscopic Analysis of Dcp1/Dcp2 Phase Separation
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Microscopy images were collected on an inverted widefield fluorescence Nikon Ti-E microscope equipped with a Hamamatsu Flash4.0 camera using either PlanApo 20x or 40x air objectives. Samples were imaged in a Greiner Bio-One 384-well glass bottom plate PEGylated using 20 mg/mL PEG-Silane (Laysan Bio, MPEG-SIL-5000) and passivated with 100 mg/mL BSA as described (Keenen et al., 2018) (link). Prior to addition of samples, the wells were washed 3x with 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT. Dcp1/Dcp2 constructs assayed by microscopy for phase separation were prepared by initiating removal of the N-terminal MBP solubility tag with 1:40 molar equivalent TEV:Dcp1/Dcp2. Dcp1/Dcp2/Edc3 droplets were prepared by incubating Dcp1/Dcp2 and Edc3 prior to removal of the N-terminal MBP tag from Dcp1/Dcp2ext. TEV cleavage and droplet settling were allowed to occur for 30 minutes at room temperature prior to imaging. For localization and enrichment of Dcp1/Dcp2ext, Edc3, or RNA in droplets, samples were prepared so that 5% of a given component was fluorescently labelled.
Image analysis was performed in FIJI (Schindelin et al., 2012) (link).
Image analysis was performed in FIJI (Schindelin et al., 2012) (link).
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