Common Buffers. PHEM (500 mls) 2x g Pipes 6.5 g Hepes 3.8 g EGTA 0.99 g MgSO 4 ph 7.0 w/ KOH

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1 Common Buffers PHEM (500 mls) 2x g Pipes 6.5 g Hepes 3.8 g EGTA 0.99 g MgSO 4 ph 7.0 w/ KOH PBS (5x in 500 mls) g NaCl g KCl g Na 2HPO 4*7 H 2O g KH 2PO 4 ph 7.2 Mounting Media 20mM Tris ph % N-propyl gallate 90% Glycerol Store at 4oC PM Buffer* 100 ml 1 M PIPES (100 mm PIPES ph= 6.9) 2 ml 1 M MgSO 4, (2 mm MgSO 4) 2 ml 0.5 M EGTA (1 mm EGTA,) distilled water to 900 ml adjust ph to 6.9 Valap* Put 50 g Vaseline, 50 g lanolin, and 50 g paraffin (all available from Fisher) in a 1 L Pyrex beaker Heat on "low" setting on a hotplate, stirring occasionally, until all components are melted and well mixed Pour into several small screw-cap jars (~50 ml capacity) 20 x Energy Regeneration System* 150 mm creatine phosphate (Boehringer #127574) 2 ml of 100 mm MgATP Stock (20 mm ATP, 20 mm MgSO 4) 40ml 0.5 M EGTA (2 mm EGTA) Disburse into 100 ml aliquots and store at -20oC

2 Isolation Buffer* 20 ml 5 M NaCl Stock (0.1 M NaCl) 4 ml of 1 M MgSO 4 Stock (4 mm MgSO 4) 2 ml of 0.5 M EDTA (1mM EDTA) 10 ml of 1 M HEPES Stock (10 mm HEPES) distilled water to 900 ml adjust ph to 7.0 High Salt Buffer* 120 ml of 5 M NaCl Stock (0.6 M NaCl) 4 ml of 1 M MgSO 4 Stock(4 mm MgSO 4) 2 ml of 0.5 M EDTA (1mM EDTA) 10 ml of 1 M HEPES Stock (10 mm HEPES) distilled water to 900 ml adjust ph to x Column Buffer* 500 ml 1 M PIPES Stock (250 mm PIPES) 40 ml of 0.5 M EGTA Stock (10 mm EGTA) 40 ml of 1 M MgSO 4 Stock (5 mm MgSO 4) distilled water to 1800 ml adjust ph to 6.7 distilled water to 2 liters Store at 4oC Homogenization Buffer* 300 ml PM buffer 52.3 mg PMSF (1mM PMSF) 3 g leupeptin (10 mg/ml leupeptin) 300 mg pepstatin A (1 ug/ml pepstatin A) 3 mg TAME (10 ug/ml TAME) mix well to dissolve, use immediately PMG Buffer* 80 ml 1 M PIPES Stock (80 mm PIPES) 2 ml 1 M MgSO 4 Stock, (2 mm MgSO 4) 2 ml 0.5 M EGTA Stock (1 mm EGTA,) 600 ml Glycerol distilled water to 900 ml, mix well adjust ph to 6.9

3 PBS* 8.18 g NaCl (140 mm NaCl) g KCl (2.5 mm KCl) g KH 2PO 4 (1.6 mm KH 2PO 4) 2.15 g Na 2HPO 4 (15 mm Na 2HPO 4) 1M MgSO 4 Stock* g MgSO 4 distilled water to 200 ml 1 M HEPES, ph = 7.0 Stock* g HEPES (free acid) distilled water to 400 ml add solid NaOH a few pellets at a time while mixing until the ph is ~6.8 add concentrated NaOH dropwise to achieve ph = 7.0 distilled water to 500 ml sterile filter and 1 M PIPES, ph = 6.9 Stock* g PIPES (free acid) distilled water to 400 ml add solid NaOH a few pellets at a time while mixing until the ph is ~6.7 add concentrated NaOH dropwise to achieve ph = 6.9 distilled water to 500 ml sterile filter and 0.5 M EDTA Stock* g EDTA (Sodium Salt) distilled water to 90 ml Adjust ph to 7.0 Distilled water 100 ml 0.5 M EGTA Stock* g EGTA (Sodium Salt) distilled water to 90 ml Adjust ph to 7.0 Distilled water to 100 ml

4 1M dithiothreitol (DTT)* g dithiothreitol disburse into 500 ml aliquots and store at -20oC 1 M KCl Stock* g KCl 10 M NaOH Stock* 40 g NaOH distilled water to 100 ml 100 mm MgGTP Stock* check formula weight of the lot of GTP you have, determine the amount required for 10 ml of a 100 mm solution. add 8.5 ml distilled water to the determined amount add 1 ml of 1M MgSO 4 Stock adjust ph to 7.0, disburse into 200 ml aliquots, store at -20oC 100 mm MgATP Stock* check formula weight of the lot of ATP you have, determine the amount required for 10 ml of a 100 mm solution. add 8.5 ml distilled water to the determined amount add 1 ml of 1M MgSO 4 Stock adjust ph to 7.0 disburse into 200 ml aliquots, store at -20oC 5 M NaCl Stock* g NaCl 3.6 mm Brefeldin A* 10 mg Brefeldin A (available from Epicenter Technologies Madison, WI) absolute ethanol to 12 ml store at -20oC

5 30 x NaF* 378 mg NaF (0.9 M NaF) Store at -20oC 30 x AlCl 3* 20 mg (1.5 mm AlCl 3) distilled water to 100 ml 30 mm Mg GTP-g-S* 10 mg GTP-g-S tetralithium salt (available from Boehringer Mannheim # ) 18 ml 1 M MgSO 4 Stock (30 mm MgSO 4) add 572 ml distilled water (to 592 ml) Store at -20oC 150 mm Mg AMP-PNP* 25 mg AMP-PNP (available from Boehringer Mannheim # ) 90 ul 1 M MgSO 4 Stock (150 mm MgSO 4) add 225 ml distilled water (to 315 ml) Store at -20oC 100 mm Sodium orthovanadate* g Sodium orthovanadate distilled water to 8 ml in a screw cap tube adjust ph to 10 if solution is yellow, place in boiling water until clear recheck ph, repeat as necessary adjust to final concentration by checking A265 nm, ext. coeff.= 2925M-1cm-1and adding distilled water as needed store at -20oC 3 M KI* 4.98 g KI add 0.5 M Na 2CO 3* 531 mg Na 2CO 3

6 add L B Broth In 1 liter of ddh 2O, combine: 10 grams Bacto-tryptone 5 grams Bacto-yeast Extract 5 grams NaCl Stir until dissolved. *=As published in... Waterman-Storer, C.M. (In press) Microtubule/organelle motility assays. In: Current Protocols in Cell Biology, J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott-Schwartz, and K.M. Yamada, eds. John Wiley, NY.