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LABELING TUBULIN 

Materials:
  • 60 mg Cycled Tubulin™ (Cat. No. 032005)
  • 5 mg NHS Ester Dye
  • Tubulin PEM Buffer (Cat. No. 032002; 80 mM PIPES, 1 mM EGTA, and 1 mM MgCl2, pH = 6.8)
  • 5X PEM Buffer (400 mM PIPES, 5 mM EGTA, and 5 mM MgCl2 , pH = 6.8)
  • High pH Cushion (100 mM Na-HEPES pH 8.6, 1 mM EGTA, 1 mM MgCl2 , 60% v/v glycerol)
  • Low pH Cushion (60% v/v glycerol in Tubulin PEM Buffer)
  • Labeling Buffer (100 mM Na-HEPES pH 8.6, 1 mM EGTA, 1 mM MgCl2 , 40% v/v glycerol)
  • Quench Buffer (2X PEM Buffer, 100 mM K-Glutamate, 40% v/v glycerol)
  • IB (50 mM K-Glutamate, 0.5 mM MgCl2 )
  • MgCl2
  • GTP
  • Glycerol
  • Liquid Nitrogen
Equipment:
  • Floor ultracentrifuge and rotor (i.e. Beckman Optima L-90K Ultra with Ti 50.2 rotor)
  • Tabletop ultracentrifuge and rotor (i.e. Beckman TL-100 Ultra with TLA-100.3 rotor)
  • Water bath at 37°C
  • Spectrophotometer
Technical Notes:
  • Tubulin must be labeled in its polymerized form to protect polymerization competency
  • Avoid diluting tubulin beyond its critical concentration
  • Work in a 37°C water bath when tubulin is polymerized, work in an ice bucket when tubulin is depolymerized
  • Pre-warm/chill necessary buffers, centrifuges, and rotors as needed
  • Dye should be used fresh from anhydrous stock
Protocol:
1. Polymerize

a. Combine GTP at 1 mM, MgCl 2 at 3.5 mM, and 60 mg Cycled Tubulin™ at 10 mg/ml in 0.5X Tubulin PEM Buffer

b. Incubate on ice for 5 minutes

c. Incubate in a 37°C water bath for 2 minutes

d. Add 1/2 volume of pre-warmed glycerol

e. Incubate in a 37°C water bath for 30 minutes


2. Pellet and Concentrate Microtubules

a. Layer polymerized microtubules over a pre-warmed High pH Cushion

*cushion should be roughly twice the volume of the polymerization reaction

b. Spin at 40,000 rpm for 45 minutes at 37°C in a pre-warmed ultracentrifuge and rotor

c. Wash the supernatant/cushion interface three times with pre-warmed Labeling Buffer

*work in a 37°C water bath with cut pipet tips to prevent MT depolymerization

d. Pipet off remaining cushion

e. Gently wash the microtubule pellet with pre-warmed Labeling Buffer

f. Resuspend the microtubule pellet in a minimal amount of pre-warmed Labeling Buffer

g. Make homogenous by pipetting up and down for 5 minutes in a 37°C water bath


3. Label Microtubules

a. Resuspend 5 mg NHS Ester Dye in DMSO

b. Add dye to microtubules in ~15 molar excess

c. Incubate in a 37°C water bath for 40 minutes with occasional mixing by pipet

d. Add equal volume Quench Buffer and mix by light vortexing

e. Incubate in a 37°C water bath for 5 minutes


4. Pellet Labeled Microtubules

a. Layer labeled microtubules over a pre-warmed Low pH Cushion

*cushion should be roughly twice the volume of the polymerization reaction

b. Spin at 80,000 rpm for 20 minutes at 37°C in a pre-warmed ultracentrifuge and rotor

c. Wash the supernatant/cushion interface three times with pre-warmed Tubulin PEM

*work in a 37°C water bath with cut pipet tips to prevent MT depolymerization

d. Pipet off remaining cushion

e. Gently wash the microtubule pellet with pre-warmed Tubulin PEM Buffer


5. Depolymerize

a. Resuspend the microtubule pellet in a minimal amount of ice-cold IB

b. Incubate on ice for 25 minutes while continuously homogenizing with a dounce


6. Clarify

a. Spin at 80,000 rpm for 10 minutes at 4°C in a pre-chilled ultracentrifuge and rotor

b. Collect the supernatant on ice


7. Cycle the Labeled Tubulin: Polymerize

a. Add GTP to 1 mM and MgCl2 to 4 mM

b. Incubate on ice for 5 minutes

c. Incubate in a 37°C water bath for 2 minutes

d. Add 1/2 volume of pre-warmed glycerol

e. Incubate in a 37°C water bath for 30 minutes


8. Cycle the Labeled Tubulin: Pellet Microtubules

a. Layer polymerized microtubules over a pre-warmed Low pH Cushion

*cushion should be roughly twice the volume of the polymerization reaction

b. Spin at 80,000 rpm for 20 minutes at 37°C in a pre-warmed ultracentrifuge and rotor

c. Wash the supernatant/cushion interface three times with pre-warmed IB

*work in a 37°C water bath with cut pipet tips to prevent MT depolymerization

d. Pipet off remaining cushion

e. Gently wash the microtubule pellet with pre-warmed IB


9. Cycle the Labeled Tubulin: Depolymerize

a. Resuspend the microtubule pellet in a minimal amount of ice-cold IB

b. Incubate on ice for 30 minutes with occasional mixing by pipet


10. Cycle the Labeled Tubulin: Clarify

a. Spin at 80,000 rpm for 10 minutes at 4°C in a pre-chilled ultracentrifuge and rotor

b. Collect the supernatant on ice


11. Measure Tubulin Concentration and Labeling Stoichiometry

a. Dilute a sample 1:150 in IB and measure A 280 and A max

b. Calculate the following using the dilution factor (DF=150), tubulin extinction coefficient (EF T = 115,000), dye extinction coefficient (EF D), and dye correction factor (CF)

i. [tubulin] = (OD 280 – (OD max * CF)) * DF / EF T

ii. [dye] = OD max * DF / EF D

iii. Labeling Stoichiometry = [dye] / [tubulin]


12. Store

a. Flash freeze in liquid nitrogen and store at -80°C

*aliquot so as to avoid repeated freeze-thaw cycles