Staining of Tau protein aggregates with Heparin

Phase-separated droplets were prepared using Tau protein and Heparin. Thioflavin T or Congo Red was added at the time of preparation.

【Sample conditions】
Tau-441 （2N4R） WT：10 μmol/l
Heparin：20 %
Buffer： 10 mmol/l HEPES （pH 7.4） 150 mmol/l NaCl
Dye concentration：10 μmol/l
Incubation time（37℃） ：10 days

【Detection conditions】
Confocal Laser Scope:　Zeiss LSM800
・Thioflavin T Ex=488 nm, Em=500-600 nm
・Congo Red Ex=561 nm, Em=550-700 nm
　Scale bar： 10 μm

Staining of BSA proteins with the crowding agent PEG8000

Phase-separated droplets were prepared using bovine serum albumin (BSA) included in LLPS Starter Kit （LL01） and the crowding agent PEG8000. ANS or SepaFluor was added at the time of preparation.

【Sample conditions】
BSA：150 μmol/l
PEG8000：15 %
Buffer： 50 mmol/l HEPES （pH 7.4）150 mmol/l NaCl
Dye concentrations：ANS 10 μmol/l SepaFluor 100 nmol/l
Incubation time（25℃）：1 hour

【Detection conditions】
Confocal Laser Scope:　Zeiss LSM800
・ANS Ex=405 nm, Em=400-550 nm
・SepaFluor Ex=640 nm, Em=650-700 nm
　Scale bar： 10 μm

Staining of Lactoferrin protein with PEG8000

Experiments were performed using Lactoferrin protein, which is known to form phase-separated droplets mainly through electrostatic interactions. LLPS Forming Condition Screening Kit （LL02） was used to investigate the concentration of PEG8000 and as the concentration was increased, the phase separated droplets of lactoferrin became gelatinous. This change was detected by SepaFluor.

【Sample conditions】
Lactoferrin：20 μmol/l
PEG8000：5 % or 10 %
Buffer： 20 mmol/l HEPES （pH 7.4）
Dye concentration：SepaFluor 100 nmol/l
Incubate time（25℃）：1 hour

【Detection conditions】
Confocal laser scopie:　Zeiss LSM800
・SepaFluor Ex=640 nm, Em=650-700 nm
　Scale bar： 10 μm