SOLUTIONS

2x MBS

50mm MES sodium salt 10.86g/l
300mm NaCl 17.53g/l

pH to 6.5 with HCl

1% Triton Lysis Buffer

dilute 50ml 2xMBS +49 water +1ml TritonX-100
on day of exp't add protease inhibitors for 5ml

NaF 1M 5ul
vanadate 100mM 100ul
aprotinin 10mg/ml 10ul
leupeptin 5mg/ml 10ul
PMSF .33M 15.15ul

take amount for 80% sucrose 0.8g sucrose/ ml eg 6 gradients x 0.8= 4.8gm sucrose to 6ml in 1% triton in MBS + protease inhibitors

5% Sucrose Stock

5gm sucrose /100ml 1xMBS, filter sterilize. Store at 4oC

on day of exp't
take amount (5ml/gradient) required for exp't and add protease inhibitors

Na vanadate 100mM 100ul /10ml
PMSF .33M 3ul/10ml

30% Sucrose Stock

30gm sucrose in 50ml 2xMBS bring up to 100ml with water, filter sterilize. Store at 4oC

on day of exp't
take amount (5ml/gradient) required for exp't and add protease inhibitors
Na vanadate 100mM 100ul /10ml
PMSF .33M 3ul/10ml

-lyse cells to a max of 1ml in lysis buffer
-homogenize using approx 50-60 strokes in dounce homogenizer
-put 1ml in ultracentrifuge tube (Beckman Ultra-Clear centrifuge tube # 344059, 50/pkg) and add 1ml of 80% sucrose
mix
-pour gradient with gradient former (Labconco Auto-densi Flow fractionator) 4.7ml of 5% sucrose and 4.7ml of 30% sucrose
-weigh each tube and balance
-run overnight(approx 15-17hr) at 37000rpm, 4oC, zero deceleration in a SW41.1 swinging bucket rotor.
-mark lipid raft fraction boundary, aspirate to upper mark and pipet off fraction and place into tube ( approx 1ml) then add 9.5ml 1xMBS with protease inhibitors, mix well and pour into an ultracentrifuge tube.
-balance and centrifuge at 37000rpm at 4oC for 1hr with deceleration at 8, in a SW41.1 swinging bucket rotor.

SDS-PAGE for Protein

back to original tube from initial run
- take 200ul from 40% sucrose (from just above the pellet) and add 200ul 2x sample buffer(TS), place on ice
-aspirate remaining liquid and wash pellet with 500ul 1xMBS and aspirate
-resuspend pellet on 200ul 2xsample buffer and place pellet in tube
-add another 300ul to tube.(TI)
-freeze thaw TI tube 3x in EtOH/dry ice & 5min 98oC then run thru a 25G needle

lipid raft fraction (LR)
aspirate all and dry tube with cutex, add 50-100ul 2xsample buffer and place in tube.
Boil for 3min and freeze at 20oC

2x sample buffer 5ml

200ul 1M DTT
4.8ml 2x Laem