Preparation of mouse liver microsome

Authors：

Category

Biosynthesis & Metabolism

Protocol Name

Authors

Haga, Yoshimi
Glycometabolome Team, RIKEN Global Research Cluster

KeyWords

Reagents

●	Buffer A (200 mM sucrose, 50 mM HEPES-KOH (pH 7.5), 40 mM KOAc, 5 mM MgCl₂, 1 mM EDTA, 1 mM DTT and protease inhibitors)
●	Buffer B (200 mM sucrose, 50 mM HEPES-KOH (pH 7.5) and 1 mM DTT)
●	1 M dithiothreitol (DTT) in water
●	Protease inhibitors (Complete protease inhibitor mixture tablets; Roche Applied Science, Penzberg, Germany)
●	1.2 M sucrose in buffer A
●	50 mM EDTA in buffer B
●	0.5 M sucrose in buffer B

Instruments

●	Motor-driven Potter-Elvehjem homogenizer (AS ONE Corporation, Osaka, Japan)
●	Dounce homogenizer
●	Dissecting instruments (scissors, tweezers, etc.)
●	Razor blade
●	Centrifuge (Avanti HP-30I, Beckman Coulter, Inc. Brea, CA)
●	Ultracentrifuge (swing rotor) (himac CS100GXL, Hitachi, Ltd., Tokyo, Japan)
●	Absorptiometer (NanoDrop, Thermo Fisher Scientific Inc., Waltham, MA)

Methods

Preparation of crude rough microsomes

1)	Sacrifice mice and remove the livers with scissors.	Comment 0

2)	Immediately rinse repetitively with buffer A. All subsequent steps have to be carried out at 0–4ºC.	Comment 0

3)	Mince the livers with a razor blade.	Comment 0

4)	Add 4 mL of buffer A per gram of liver.	Comment 0

5)	Homogenize with 5 strokes (10 sec down, 10 sec up) in a motor-driven Potter-Elvehjem homogenizer.	Comment 1

6)	Centrifuge the homogenate for 10 min at 1,000 × g.	Comment 0

7)	Remove floating fatty materials and collect the supernatant.	Comment 0

8)	Centrifuge for 10 min at 10,000 × g.	Comment 0

9)	Immediately collect the supernatant.	Comment 1

10)	Ultracentrifuge for 2.5 h at 140,000 × g through a cushion of 1.2 M sucrose in buffer A. (The ratio of load to cushion: 3:1)	Comment 0

11)	Remove the supernatant, including the cushion, by aspiration.	Comment 0

12)	Resuspend the pellet by Dounce homogenizer (2–3 strokes) in buffer B to a concentration of 50 A₂₈₀ units/mL (solubilized in 1% SDS solution when determined). In a typical preparation, 50 A₂₈₀ units of rough microsomes are obtained from 1 g tissue.	Comment 0

13)

The microsomes may be flash frozen in liquid nitrogen and stored at –80ºC until use.

Comment 0

EDTA stripping of membrane-bound ribosomes (Optional)

1)	Mix rough microsomes and 50 mM EDTA in buffer B at a rate of 1:1.	Comment 0

2)	Incubate for 15 min at 0–4ºC.	Comment 0

3)	Centrifuge the mixture for 1 h at 140,000 × g through a cushion of 0.5 M sucrose in buffer B. (The ratio of load to cushion: 3:1)	Comment 0

Resuspend the pellet in same volume of buffer B.

Comment 0

Notes

Buffers are recommended to be filtrated through a 0.45 μm Millipore filter.
1 M DTT stock solution was kept in small aliquots at −20ºC and diluted into the buffers immediately before use.
Protease inhibitors were also kept in small aliquots at −20ºC and diluted into the buffers immediately before use.

Initial amount

Livers from four mice: 1.5 g.

Produced amount

1–2 mL microsomal fraction

Copyrights

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Date of registration：2014-12-18 13:43:09