You can also find the protocol at: https://snownontrace.github.io/microtubule-co-sedimentation-worm-extracts.html

Microtubule co-sedimentation from worm extracts

Author: Shaohe Wang Date: 2021-04-07

Please use detailedprotocols described in this paper to make large amount of worm materialthat has been drop frozen in liquid nitrogen as frozen droplets ("worm beads") stored at -80°C.
Prepare or purchase buﬀers and reagents, including:

○ 1M 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 storeat -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 afteradding 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 dissolvethe 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

Stock	for 10 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 dissolvethe 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

4. 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-waterbath 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 (22,000rpm) 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 determinethe 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.

5. (Optional) Make exogenous taxol stabilized microtubules.

○ Onice, 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 37°C for 1-2 min to polymerize microtubules.

○ Add taxol stepwise to equimolar as follows (for 2 mg/ml tubulin). For each step, pipetin 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.

6. 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++).

○ Onice, 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 carefullyremoving 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.

7. 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.