Membrane Floatation Experiments: (beth 1/02)

homogenize two rat brains or other cells, tissue etc. in buffer - I used PHEM, but should try Schnapp buffer.

Low speed spin at 12 k for 30 min.

High speed spind of sup - 36 k for 1 hour.

Resuspend pellet in 300 ul of buffer with DTT and protease inhibitors. Probably need to use small homogenizer.

Divide membrane into different fractions - ie. membranes + buffer, membrane + slat, etc.

incubate at room temp for 15-20 min.

Mix each 200 ul sample with 800 ul of 2.5 M sucrose. Place in the bottom of a 12 ml swinging bucket centrifuge tube.

Layer onto this sample 6 ml of 1.5 M sucrose, and then 5 ml of 0.6 M sucrose.

Centrifure at 40 K for 2 hours at 4 C. (Maybe try 3-4 hr with spectrin to clarify 1.5 M layer - i saw a lot of trailing in this

fraction which I didn't see with dynein)

Collect fractions from 0.6/1.5 M interphase, and from the 1.5M and 2 M fractions.

That's the basic prep. I tried extractions with KI and neomycin like Schnapp did, but nothing was extracted in PHEM. That's why I suggest

the Schnapp 1/2X buffer.