Phase separation assays - Method

Microscopic LLPS assay

protocol: with fluorescent RNA - “LLPS assay - Niklas Schaub.docx”

protocol: fluorescentHisBinder-N - XXXXstill missing

overview to RNA oligos for LLPS: RNA oligos - Nucleocapsid project

overview to long RNA molecules for LLPS: XXXXX

Turbidity assay 350nm Nanodrop, Julia Welte

Assay acc. to (Zavastano Zweckstetter 2020 PMID: 33247108)

Buffer: !? apparently 20 mM NaPi, pH 7.5 without salt

Protein: 117a (40uM) - (20 mM Tris ph8 50mM NaCl + 10% Glycerol)

RNA PolyU (10mg/ml in water; mean MW 1000kDa; 10 uM)

Assay in 10 ul: 117a 20uM; RNA variable conc.; fill up with buffer

Measure in Nanodrop

Optimisation of LLPS turbidity assay - Niklas Schaub

1. Lowering salt - (Carlson MollCell 2020 PMID: 33248025) The day prior to imaging, protein was thawed and dialyzed against droplet buffer (25 mM HEPES pH 7.5, 70 mM KCl) overnight at 4°C. Protein concentration was quantified by nanodrop.
   According to (Zavastano 2020 PMID: 33247108) probably even better lower - 0mM.
2. Crowding agents - (Luo 2021 - strange chinese journal PMID: 33495715) 2% Ficoll - LLPS from 15uM to 2uM N.
   (Chen CellRes 2020 PMID: 32901111) 2.5% PEG8000
3. Buffer - (Chen CellRes 2020 PMID: 32901111) Zn2+ 20uM had a significant effect on enhancing the LLPS
4. blocking surface of 384 well plate . Use Pluronic + PEG solution. Wash with Buffer. in case it is required.