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Turbidity assay

AG Abner Gonzalez
TM Taro Mannen
TÇ Tolga Çağatay
AF Ayano Fujiwara
HM Hiroyoshi Matsumura
AN Ashley B. Niesman
CB Chad A. Brautigam
YC Yuh Min Chook
TY Takuya Yoshizawa
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Prior to adding Tev protease, MBP-FUS, ± Importinβs, ± RanGTP, ± Importinα IBB(residues 1–74), ± M9M peptide were mixed in buffer containing 20 mM HEPES pH 7.4, 150 mM NaCl, 2 mM magnesium acetate, 20 μM zinc acetate and 2 mM DTT with 10% glycerol. Tev protease was added to the premixture, to a final concentration of 25 μg/mL then incubated at room temparature for 60 min. Absorbance at 395 nm (OD395 nm) was measured using plate reader. For the Kapβ2 titration turbidity assay, Tev digestion for 8 uM MBP-FUS was initiated in the absence or presence of 2–16 uM Kapb2 at 30 °C. After 1 h, the samples cooled down to 10 °C then measured OD395 nm. All experiments were performed three or four technical repeats and represented as mean ± SD.

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