Isolation of Sf21/Sf9 microsomes (without clumping)
jma at ddt.biochem.umn.edu
Tue Nov 29 16:52:41 EST 2005
I am fractionating baculovirus-infected Sf9 and Sf21 insect cells, using differential centrifugation to isolate microsomal membranes.
The problem is that the microsomes (50,000 x g) are very particulate, and remain clumpy even after homogenization with hand-held glass/Teflon Potter-Elvehjem tissue grinder. Even mechanical disruption of the final microsomal pellet using a small polytron leaves clumps of unresuspended membranes that are visible by eye.
The insect cell microsomes are clumpy when isolated either in solutions containing high salt (0.6 M KCl, 250 mM sucrose, 3 mM MgCl2) or low salt (10 mM NaHCO3, 0.1 mM CaCl2). The two different preparative protocols are listed below.
Following centrifugation, the microsomal pellets are typically resuspended in 250 mM sucrose buffered with MOPS or histidine.
Why do the microsomes clump so much? Are there solutes to add (pyrophosphate, NaI, etc) that would allow easy and complete resuspension of insect cell microsomes?
Research Associate with Dave Thomas
Virus-infected Sf21 cells in 600 ml of suspension (9 × 108 cells) were sedimented, washed twice with phosphate-buffered saline, and resuspended in 50 ml of medium containing 10 mM NaHCO3, 0.2 mM CaCl2, plus the following protease inhibitors: aprotinin (10 µg/ml), leupeptin (2 µg/ml), pepstatin A (1 µg/ml), and Pefabloc (0.1 mM), which were included throughout the entire preparative procedure. Cells were disrupted by N2 cavitation using a Parr Cell Disruption Bomb 4635 (Parr Instruments, Moline, IL), and cellular homogenates were diluted into an equal volume of ice-cold medium containing 500 mM sucrose, 300 mM KCl, 6 mM MgCl2, and 60 mM histidine (pH 7.4). Homogenates were centrifuged at 1000 × g for 20 min. Supernatants were recovered, diluted with 0.25 volume of 3 M KCl, and centrifuged at 10,000 × g for 20 min. Supernatants were again recovered and centrifuged at 50,000 × g for 30 min. Pellets were washed in 250 mM sucrose, 600 mM KCl, 3 mM MgCl2, 30 mM histidine (pH 7.4) and sedimented as before. Final pellets (Sf21 microsomes) were resuspended at approximately 5 mg/ml in 250 mM sucrose, 30 mM histidine (pH 7.4) and stored in small aliquots at -40 °C. The average yield per 600 ml of infection was 40 mg of microsomal protein.
Low salt protocol:
For preparation of Sf21 cell microsomes, the washed cell pellets were resuspended in 25 ml of medium containing 10 mM NaHCO3 and 0.2 mM CaCl2, supplemented with the protease inhibitors aprotinin (1 µg/ml), leupeptin (0.2 µg/ml), pepstatin A (1 µg/ml), and Pefabloc SC (0.1 mM). The cells were transferred to JA-20 centrifuge tubes and homogenized with a Polytron three times for 30 s at 14,000 rpm with 10-s breaks. The homogenates were centrifuged at 12,000 × g for 45 min in a Beckman JA-20 rotor, and the supernatants were collected and spun in a Beckman type 70 Ti rotor at 50,000 × g for 35 min. The pellets were resuspended in 1.5-2 ml in 50 mM MOPS, 0.3 M sucrose, and 5 mM NaN3 (pH 7.0) supplemented with the protease inhibitors above and subjected to a final homogenization in a 2-ml Potter-Elvehjem tissue grinder.
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