A. Ca and Mg free Phosphate Buffered Saline (PBS) solution, buffered with 0.02M Hepes. pH=7.4
B. Ca and Mg free PBS, buffered with 0.02M Hepes, and 1mM EDTA. pH=7.4
C. PBS with 4% Bovine Serum Albumin (BSA) (weight/ vol)
D. PBS, plain.
E. 0.01M Tris HCl. pH=7.4
F. 140mM NaCl, 10mM Tris Buffer
G. Sucrose Gradient: 60% sucrose and 3.0mM MgCl in 0.01M Tris HCl. pH=7.4, 20% sucrose, 10% sucrose (wt/vol in 0.01M Tris HCl).
A. Cell treatment and collection
Farm L929 cells on 150mm plates (10 plates yeilds approx. 550ug of protien).
Treat cells with latex beads (Polybead polystyrene microspheres 4.55 x 10 beads/ml-- Polysciences Inc.) for 1-2hrs prior to harvesting. Use 2,000 beads per cell and add directly to the media-- media will turn a cloudy pink.
After the bead treatment, aspirate off the media and wash 1x with 10-20mls of -Ca-Mg PBS buffered with 0.02M Hepes.
Harvest cells by putting 20mls of -Ca-Mg PBS, buffered with 0.02M Hepes, and 1mM EDTA on each plate for 10min in the 37 C incubator.
After the 10min, collect some of the PBS in a 50ml conical tube before pipeting up and down to remove cells. Collect cells in the same 50ml tube as the PBS.
Once cells are collected, spin at 4C at 150 x g for 10min (Clinical centrifuge in cold room, setting #3).
All following steps done at 4C. Also, pool samples so that working with a maximum of 4 50ml conical tubes.
While cell samples are spinning, put 20mls of the 4% BSA PBS into new 50ml conical tubes. When samples are finished spinning, resuspend the pellet in 25mls of PBS. Transfer the sample to the tubes with the 4% BSA PBS. Spin at 4C at 300 x g for 15min (Clinical centrifuge in cold room, setting #4).
Wash 2x's with PBS, spin at 4C at 150 x g for 5min for each wash.
Resuspend the pellet in 2.5mls of 0.01M Tris HCl, pH 7.4. Let the samples sit on ice for 3min.
Pool two samples and place in a tight-fitting Dounce Homogenizer.
Homogenize vigorously for approximately 15min. Check periodically to see if cells are sheared by using a hemocytometer. (Cells should go from round and glowing to looking like debris).
Take out about 2mls at this point for alkaline phosphatase assay.
Add 5mls of the 60% sucrose to the 5mls of homogenate (this reduces the 60% sucrose to 30% sucrose for the gradient).
C. Sucrose Gradient and Centrifugation
Put 3.330mls of the homogenate-30% sucrose mixture into 3 Beckman ultra clear centrifuge tubes-- can only use a maximum of six tubes for this particular spin.
Repeat the homogenization with the other 2 samples.
Once all the samples are in the ultra clear tubes the other sucrose solutions can be added to complete the gradient. By using a 10ml syringe with a 5cc 22G 1 1/2 needle add 6mls of the 20% sucrose and 3mls of the 10% sucrose to each tube.
Place the tubes in the SW41 rotor buckets and weigh before spinning. Spin in the ultracentrifuge at 4C at 110,000 x g (30,000 RPM) for 90min.
After spinning, collect fractions by using a tube slicer. Desired fraction (membrane fraction) is between the 10% and 20% sucrose layers, collect and place in separate ultra clear tubes. All other fractions can be pooled into 15ml conical tubes and stored at -70C.
Resuspend the desired fraction in 12mls of 140mM NaCl and 10mM Tris. put in SW41 rotor buckets and weigh. Spin in the ultracentrifuge at 4C at ? x g (8000 RPM) for 30min.
After spinning, aspirate off the buffer and resuspend the pellet in 500ul of 10% sucrose. Store samples in cryoviles as 200ul aliquots in -70C.
Do a protein assay and an alkaline phosphotase assay.
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