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Homogenization buffer (HB)

AM Arpan G. Mazumder
AJ Amélie M. Julé
DS Daniel Sun
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The homogenization buffer was prepared by adding 2.5 mL of 2 M KCl (RNase free; Fisher Scientific, #AM9640G), 2.5 mL of 1 M Tris (pH 7.4; ThermoFisher Scientific, #17,926), 500 µl of NP-40 (ThermoFisher Scientific, #28,324) and 600 µl of 1 M MgCl2 (RNase free; Fisher Scientific, #AM9530G) into a 50 mL Falcon tube. The volume was adjusted to 50 mL with DNase/RNase free water and vortexed until all contents were completely dissolved. The supplemented homogenization buffer (HB-S) was always prepared fresh prior to homogenization by adding 50 µl of protease inhibitor (VWR, #97,063–970), 5 µl of 1 M DTT (VWR, #97,061–340), 25 µl of RNasin (VWR, #PAN2615), 50 µl of Heparin (100 mg/ml; Fisher Scientific, #BP252420) and 100 µl of cycloheximide (5 mg/ml; Fisher Scientific, #AC357420050) in a 15 ml Falcon tube. Finally, the volume was adjusted to 5 mL with HB buffer.

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