Hydrogels of Cytoskeletal Proteins by Yoshihito Osada Ryuzo Kawamura & Ken-Ichi Sano

Author:Yoshihito Osada, Ryuzo Kawamura & Ken-Ichi Sano
Language: eng
Format: epub
Publisher: Springer International Publishing, Cham

2.Homogenize the brain using a blender (at 7800 rpm, T25 digital, IKA) for 3 min after adding ice-cold depolymerization buffer (DB) [50-mM 2-(N-morpholino)ethanesulfonic acid (MES), 1-mM CaCl2, 0.1-mM GTP, protease inhibitor (Complete EDTA-free, 11873580001, Roche), pH 6.6 by KOH] in a ratio of 1 mL/(1-g brain).

3.Centrifuge the homogenate 12,000 rpm (17,600 × g) at 2 °C for 60 min putting in 250-mL bottles with precooled R12A rotor (Hitachi).

4.Mix the collected supernatant with equal volume of pre-warmed glycerol and HMPB [1-M Pipes, 10-mM MgCl2, 20-mM EGTA, pH 6.9 by KOH] in 1 L Erlenmeyer flask.

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