UA Biophysics:Protocols:Tubulin Polymerization with AlexaFluor 488 staining

OBJECTIVE

The objective of this protocol is to artificially polymerize microtubules from tubulin and to stain them for fluorescence microscopy.

MATERIALS

Materials:

1. Biotin labeled tubulin
2. TAMRA Rhodamine labeled tubulin protein
3. Taxol
4. GTP [100mM]
5. BRB80 buffer
6. MgCl2 [50mM]
7. DMSO
8. AlexaFluor 488
9. PBS-BSA 0.1%
10. β-mercaptoethanol 14M
11. Wet chamber
12. Coverslips and slides
13. Nail polish

RECIPE

Microtubules were prepared by polymerizing 20 microgram of biotin-labeled tubulin (Cytoskeleton Inc., Denver, CO) in 6.5μl of growth solution containing 4 mM of MgCl2, 1 mM of GTP, and 5% DMSO (v/v) in BRB80 buffer for 30 min at 37 °C. The microtubules were then 100-fold diluted and stabilized in 10 microM paclitaxel [taxol] (Sigma, Saint Louis MO).

1. Prepare 1 ml PBS+β-mercaptoethanol 14mM. Put aside.
2. Prepare 13.0 μl of growth solution by adding
   1. i. MgCl2 [50mM]: 1.04 μl
   2. ii. GTP [25mM]: 2.00 μl
   3. iii. DMSO [100%]: 0.65 μl
   4. iv. BRB80: 9.40 μl

1. From this growth solution extract 6.5 μl and pour 20μg of biotin-labeled tubulin (Cytoskeleton Inc., Denver, CO) in it. Discard the other 6.5μl .
2. Prepare 500μl BRB80 + Taxol 10μM.
3. Disolve the 6.5 μl of growth solution + biotion-labeled tubulin in the 500μl BRB80 + Taxol 10μM.
4. From the 506.5μl take 20μl and deposit it on a thin slide.
5. Let dry.
6. Rinse with PBS-BSA 0.1% and leave for 30min at room temperature.
7. Add 20μl of Alexa 488 2000x in PBS.
8. Leave in a humid petri dish at room temperature for 30min.
9. Put a droplet of PBS+β-mercaptoethanol (~20μl).
10. Immediately put on a thick slide. Then seal with nail polish so PBS+β-mercaptoethanol does not evaporate.
11. Let dry. Do not expose to light. Ready to be imaged.