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.

WARNING

Microtubules depolymerize extremelly rapidly even in the pressence of Taxol. These samples must be imaged the same day they are prepared.

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. MgCl2 [50mM]: 1.04 μl
   2. GTP [25mM]: 2.00 μl
   3. DMSO [100%]: 0.65 μl
   4. 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.