Sucrose Density Gradient Fractionation

Grow a 2 ml culture,24 hr.at 30℃ 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 30℃

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 (421μl 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 x109 cells (about 100 O.D.equivalents (100 ml of OD = 1): 2750 X g.,10 min.,30℃ 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 -20℃)at room temp,then spin 1 min at 14,000 rpm.Take supe.-- for 4 gradients,use 900μl of supe,90μl 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,30℃

FROM HERE ONOUT KEEP EVERYTHING AT 4℃

Centrifuge 500 x g,10 min.4℃.

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.,4℃

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

Add 650μl of the sup to 606 mg (0.606 g)sucrose in a μltracentrifuge tube (this makes a 70% solution)--use tweezers to handle μltracentrifuge 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 100μl 4X SDS PAGE sample buffer

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

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

RUNNING GEL

Pre-warm samples to about 37 ℃ 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 20μl 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:

2μl/ml of beta mercaptoethanol

20μl/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)

10μg/ml leupeptin (add fresh)

10μg/ml pepstatin (add fresh)

10μg/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)