Microtubule co-sedimentation from worm extracts

Please use detailed protocols described in this paper to make large amount of worm material that has been drop frozen in liquid nitrogen as frozen droplets (“worm beads”) stored at -80°C.
Prepare or purchase buffers and reagents, including:
- 1 M Hepes-KOH (pH 7.6; use KOH for pH adjustment); store at 4°C
- 1 M Pipes-KOH (pH 6.8; use KOH for pH adjustment); store at 4°C
- 3 M KCl; store at room temperature
- 1 M MgCl₂; store at room temperature
- 0.5 M EGTA (pH 8.0); store at room temperature
- 1 M DTT; aliquot and store at -20°C
- 1 mg/mL Pepstatin A (in ethanol; may add 5% acetic acid); store at -20°C
- 100 mM GTP (Thermo Fisher, R0461); aliquot and store at -20°C
- 10 mM Taxol in DMSO (MilliporeSigma, PHL89806); aliquot and store at -20°C
- 10 mg/mL Nocodazole in DMSO (MilliporeSigma, M1404); aliquot and store at -20°C
- 4x Laemmli Sample Buffer (Bio-Rad, 1610747); add 2-mercaptoethanol before use; aliquot and store at -20°C after adding 2-mercaptoethanol

Prepare buffers.

Worm lysis buffer (50 mM Hepes-KOH, pH 7.6, 1 mM MgCl₂, 1 mM EGTA, 75 mM KCl, 0.5 mM DTT; add proteinase inhibitors before use; store on ice):

Stock	for 10 mL	for 50 mL
H₂O	9.2 mL	46 mL
1 M Hepes-KOH, pH 7.6	0.5 mL	2.5 mL
3 M KCl	0.25 mL	1.25 mL
1 M MgCl₂	10 µL	50 µL
0.5 M EGTA, pH 8.0	20 µL	100 µL
1 M DTT	5 µL	25 µL
1 mg/mL Pepstatin A	10 µL	50 µL

Add 1 tablet of protease inhibitors (MilliporeSigma, 11836170001) per 10 mL buffer. Vortex to dissolve the tablet. Store on ice.

Glycerol cushion (40% glycerol, 50 mM Hepes-KOH, pH 7.6, 1 mM MgCl₂, 1 mM EGTA, 75 mM KCl, 0.5 mM DTT; add proteinase inhibitors before use; store on ice):

Stock	for 10 mL
50% Glycerol	8 mL
H₂O	1.2 mL
1 M Hepes-KOH, pH 7.6	0.5 mL
3 M KCl	0.25 mL
1 M MgCl₂	10 µL
0.5 M EGTA, pH 8.0	20 µL
1 M DTT	5 µL
1 mg/mL Pepstatin A	10 µL

Add 1 tablet of protease inhibitors (MilliporeSigma, 11836170001) per 10 mL buffer. Vortex to dissolve the tablet. Store on ice.

BRB80 buffer (80 mM Pipes-KOH, pH 6.8, 1 mM MgCl₂, 1 mM EGTA; store on ice or at 4°C)

Stock	for 10 mL	for 1 L
H₂O	9.17 mL	917 mL
1 M Hepes-KOH, pH 7.6	0.8 mL	80 mL
1 M MgCl₂	10 µL	1 mL
0.5 M EGTA, pH 8.0	20 µL	2 mL

Make Worm Lysate.
- Weigh out ~1 g of frozen adult worm beads and add 1.5 mL of Worm Lysis Buffer (see above) in a 15 mL conical tube. The total volume is ~2.5 mL. Keep on ice.
- Use a microtip sonicator to lyse worms by sonication. Save a 100 µL CRUDE worm extract sample and keep on ice.
  - We use a Branson 450 Digital Sonifier set at 30% amplitude for 1 min total on time (0.5 s on, 0.5 s off). Usually 2-3 repeats (i.e. 2-3 min total sonication time) are needed to reach the optimal level of lysing. Samples should be chilled for at least 30 sec between sonication runs.
  - Before sonication, poke and stir the samples with a clean metal micro spatula to homogenize samples in the lysis buffer on ice.
  - Place the 15 mL tube in an ice-water bath during sonication to prevent heating. We usually use a beaker to set up the ice-water bath.
  - Monitor worm lysis using a Bradford assay by mixing a small sample (e.g., 2 µL) with 1 mL Bradford reagent. When worms are lysed the color should turn blue. Sonication can be stopped when the total protein concentration of lysate stops increase.
- Transfer crude extract to a pre-cooled centrifuge tube for the TLA100.3 rotor and spin at 20,000 g (22,000 rpm) for 10 min at 2°C. Set DECEL = 5.
- Transfer the supernatant (low speed supernatant; LSS) into a new pre-cooled centrifuge tube for the TLA100.3 rotor. Take care to avoid lipid when transferring. Repeat the centrifugation if the lysate is too cloudy. Save a 100 µL LSS sample and keep on ice.
- Spin the LSS at 50,000 g (34,000 rpm) for 20 min at 2°C.
- Transfer the supernatant (high speed supernatant; HSS) into a pre-cooled 15 mL tube on ice. Save a 100 µL HSS sample and keep on ice.
- Use a Bradford assay to determine the concentration of worm lysate. From a typical prep, the CRUDE is 18-20 mg/mL and HSS is 12-16 mg/mL.
- In a new set of tubes, mix 60 µL CRUDE, HSS and LSS samples with 20 µL 4x Sample Buffer.
(Optional) Make exogenous taxol stabilized microtubules.
- On ice, dilute stock tubulin (10 mg/mL) to 2 mg/mL in BRB80 buffer supplemented with 1 mM DTT and 1 mM GTP. Incubate on ice for 5 min.
- Clarify tubulin mix in a pre-cooled TLA120.2 rotor at 90 k rpm for 5 min at 2°C.
- Warm up a TLA100.3 rotor in a 37°C incubator.
- Transfer clarified tubulin supernatant into a new 1.5 mL tube pre-cooled on ice.
- Incubate at 37C for 1-2 min to polymerize microtubules.
- Add taxol stepwise to equimolar as follows (for 2 mg/ml tubulin). For each step, pipet in the taxol and immediately flick the tube to mix it in. If taxol is added all at once it will cause tubulin precipitation.
  - Add 1/10 vol 2 µM taxol; Incubate at 37°C for 10 min.
  - Add 1/10 vol 20 µM taxol; Incubate at 37°C for 10 min.
  - Add 1/10 vol 200 µM taxol; Incubate at 37°C for 15 min.
- Pellet microtubules over a warm 40% glycerol in BRB80 cushion in a TLA100.3 rotor at 80 k rpm for 20 min, 25°C. Put rotor back at 4°C to cool down.
- Aspirate and wash sample/cushion interface with warm BRB80 buffer.
- Rinse pellet and resuspend in warm BRB80 + 200 µM taxol. The taxol should be at least equimolar and preferably in excess to the tubulin. 2 mg/mL tubulin is ~20 µM.

Co-sedimentation of MAPs (microtubule associated proteins) with taxol-stabilized MTs.

Dilute 1 M DTT 1:10 in water to obtain 100 mM stock.
Dilute 10 mg/mL Nocodazole 1:20 in DMSO to obtain 0.5 mg/mL stock.
(Optional) Mix 100 µL BRB80 with 1 µL 100 mM DTT and 1 µL 100 mM GTP to obtain BRB80++. This is only needed as control when including the exogenous microtubule group).
On ice, supplement 5 µL 100 mM DTT and 10 µL 100 mM GTP to 1 mL HSS worm lysate (HSS++).

On ice, set up the following experimental groups. The group including exogenous microtubules is optional. If omitting this group, BRB80 is unnecessary to add. Volumes are in µL. Final concentration of supplements: 0.5 mM DTT, 1 mM GTP, 10 ug/mL nocodazole or 20 uM taxol.

Stock	no-MT-1	no-MT-2	endo-MT	exo-MT
HSS++	203	203	203	203
DMSO	0.4	-	-	-
Nocodazole 0.5 mg/mL	-	0.4	-	-
Taxol 10 mM	-	-	0.4	0.4
BRB80++	20	20	20	-
Exo-MT ~20 µM	-	-	-	20

Incubate all samples at room temperature (22~23°C) for 10 min to allow polymerization of the endogenous tubulin. The nocodazole in no-MT-2 group should prevent microtubule polymerization.
Transfer samples back to ice and incubate for 15 min. The cold temperature should depolymerize microtubules not protected by taxol in the no-MT-1 group.
In 4 pre-cooled centrifuge tubes for the TLA120.2 rotor, add 1 mL 40% glycerol cushion (see Prepare buffers) to each tube.
Carefully add the 4 mixtures prepared above (~200 µL each) onto the glycerol cushions.
Centrifuge through glycerol cushion at 48,000 g (37 k rpm) for 30 min at 4°C using a TLA120.2 rotor.
After centrifugation, take a SUPE (supernatant) sample by carefully removing 60 µL from the top and transfer into a pre-cooled 1.5 mL tube on ice. Add 20 µL 4x sample buffer.
Wash the sample/cushion interface three times. For each wash, remove the supernatant until the sample/cushion interface and add back 250 µL Worm Lysis Buffer. This washing step is to clean up the wall of centrifuge tubes that contacted the high-concentration lysate.
Aspirate the cushion and resuspend the pellet with 200 µL 1x SDS-PAGE sample buffer (PELLET).
Denature all collected samples in sample buffer for 5 min at 95°C. Store them at -20°C if not running SDS-PAGE on the same day.

Run SDS-PAGE gel(s) of CRUDE, LSS, HSS, SUPE (1-4) and PELLET (1-4) samples for Coomassie staining and/or Western blotting analysis.