SucroseDensityGradientFractionationofYeastMembranes

• Grow a 2 ml culture, 24 hr. at 30oC in selective media

• When culture is ready, use it to inoculate about 55 ml (50 ml plus 5 for O.D.600 readings) of selective media. Grow to mid-logarithmic
  phase (O.D.600 is about 1). This usually takes 10 hours (for 1ml of the 24 hr. culture into 50 ml media). It is helpful to inoculate 2
  different flasks--one with 1 ml of the 2 ml culture, the other with 0.5 ml of the 2 ml culture

• Spin at 2750 X g. for 10 min. at 30oC

• Resuspend in 100 ml pre-warmed YPD so that O.D.600 = 0.5 (50 ml of O.D.=1 culture into 100 ml total YPD)

• If appropriate, after 1 hr add 2.5 uM alpha factor (421 ul of stock (1 mg/ml) to 100 ml of culture) (we want it present during the last
  hour of growth)

• Wait 1 doubling period (about 2 hours) until O.D.600 equals about 1

• Add 1 ml of COLD 1M NaN3 to 100 ml culture (we want 10 mM total conc.)

• Spin down 3 x 109 cells (about 100 O.D. equivalents (100 ml of OD = 1): 2750 X g., 10 min., 30oC in lab clinical centrifuge

• Wash 1x with 10 ml SK buffer in 15 ml falcon tubes (see recipe below).

• Resuspend in 10 ml SK with 1 mg zymolyase and 28.8 mM Beta-mercaptoethanol to make SK:
  thaw zymolyase (kept at 5 mg/ml stock in SK at -20oC) at room temp, then spin 1 min at 14,000 rpm. Take supe.-- for 4 gradients, use 900 ul of supe, 90 ul of beta-mercaptoethanol and bring to 45 ml with SK (this recipe should be adjusted for the appropriate number of gradients--we want to conserve zymolyase--it's expensive)

• Shake gently, 45 min, 60 rpm, 30oC

FROM HERE ON OUT KEEP EVERYTHING AT 4oC

• Centrifuge 500 x g, 10 min. 4oC.

• Wash 1 x with 2 ml cold SK buffer

• Wash again with 2 ml lysis buffer C (See recipe below)

• Resuspend in 1 ml lysis buffer C and transfer to glass P-E tube (potter-elvejhem-- homogenizer tube)

• Disrupt with 25 strokes of a motorized homogenizer on ice (go entirely in and out of liquid 25X)

• Transfer to eppendorf tube, centrifuge twice (spin once then re-spin sup) at 500 X g. for 10 min., 4oC

• Remove 100 ul of sup as "input." add 100 ul 2X SDS-PAGE sample buffer. Put in 100oC heat block for 10 min., then transfer to -20oC.

• Add 650 ul of the sup to 606 mg (0.606 g) sucrose in a ultracentrifuge tube (this makes a 70% solution)--use tweezers to handle ultracentrifuge tubes, add "flea" (the tiny stir bar). Put a bunch of tubes in a small beaker and put the beaker on a stir plate in the cold room. leave at 4o until dissolved (around an hour)

• Remove flea by taking a big stir bar and putting it against the outside of the tube, moving it upwards. Gently overlay with 1 ml cold sucrose solutions of 60%, 50%, 40%, 30%

• Balance tubes with 30% sucrose solution (weigh them to confirm balance)

• Spin in swinging bucket centrifuge for 16 hr (can go longer) at 190,000 x g (37500 rpm) in a SW55Ti rotor

• Have microfuge tubes pre-labelled with 100 ul 4X SDS PAGE sample buffer

• After spin is done, collect 16 samples of 300 ul each into 100 ul of 4x SDS-PAGE sample buffer.

• Put at 100oC for 10 min, then freeze at -20oC--can store here indefinitely.

RUNNING GEL

• Pre-warm samples to about 37 oC for about 20-25 min. Vortex, spin at 14,000 for 1 min.

• Pour gel: 1.5 mm thick, 8% SDS-PAGE gel with a long stacking gel--about 1/2 of the gel should be separating, and 1/2 stacking. Also use a 15 well comb

• Load 20 ul of sup

• Run at 50 volts for about 4 hr.

• Transfer for 2 hr (or 1 hr 30 min) at 100V

SOLUTIONS

S/K (100 ml):

1.2 M sorbitol (21.84 g/100ml)
0.1 M KPO4 pH 7.5 (9.5 g dibasic (fw = 268) 1.99 g monobasic (fw = 136)

filter sterilize!

zymolyase buffer:

to S/K buffer add:
2 ul/ml of beta mercaptoethanol
20 ul/ml of zymolyase (stock solution 5 mg/ml in S/K)

spin before using!

Lysis buffer C:

20 mM TEA pH 8 
0.8 M sucrose 
1 mM EDTA 
1 mM DTT (add fresh)
1 mM AEBSF (add fresh)
10 ug/ml leupeptin (add fresh)
10 ug/ml pepstatin (add fresh)
10 ug/ml benzamidine (add fresh)

mix first 3 ingredients and filter sterilize

Sucrose:

60 g sucrose + 5 ml 200 mM TEA + H2O to 100ml for 60% solution
also prepare 50%, 40%, 30% (all also dissolved in TEA)