UA Biophysics:Protocols:Tubulin Polymerization with AlexaFluor 488 staining: Difference between revisions
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==MATERIALS== | ==MATERIALS== | ||
#Biotin labeled tubulin | #Biotin-labeled tubulin (Cytoskeleton Inc., Denver, CO) | ||
#Taxol (Sigma, Saint Louis MO) | |||
#Taxol | |||
#GTP [100mM] | #GTP [100mM] | ||
#BRB80 buffer | #BRB80 buffer | ||
| Line 17: | Line 15: | ||
#AlexaFluor 488 | #AlexaFluor 488 | ||
#PBS-BSA 0.1% | #PBS-BSA 0.1% | ||
#β-mercaptoethanol 14M | #β-mercaptoethanol [14M] | ||
#Wet chamber | #Wet chamber | ||
#Coverslips and slides | #Coverslips and slides | ||
#Nail polish | #Nail polish | ||
==RECIPE== | ==RECIPE== | ||
#Prepare 1 ml PBS+β-mercaptoethanol 14mM. Put aside. | #Prepare 1 ml PBS+β-mercaptoethanol 14mM. Put aside. | ||
#Prepare 13.0 μl of growth solution by adding | #Prepare 13.0 μl of growth solution by adding in an eppendorf | ||
## MgCl2 [50mM]: 1.04 μl | ## MgCl2 [50mM]: 1.04 μl | ||
## GTP [25mM]: 2.00 μl | ## GTP [25mM]: 2.00 μl | ||
| Line 34: | Line 29: | ||
## BRB80: 9.40 μl | ## BRB80: 9.40 μl | ||
#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 . | #From this growth solution extract 6.5 μl and pour 20μg of biotin-labeled tubulin (Cytoskeleton Inc., Denver, CO) in it. Incubate for 30min at 37°C. Discard the other 6.5μl . | ||
#Prepare 500μl BRB80 + Taxol 10μM. | #Prepare 500μl BRB80 + Taxol 10μM. | ||
#Disolve the 6.5 μl of growth solution + | #Disolve the 6.5 μl of growth solution + polymerized microtubules in the 500μl BRB80 + Taxol 10μM. | ||
#From the 506.5μl take 20μl and deposit it on a thin slide. | #From the 506.5μl take 20μl and deposit it on a thin slide. | ||
#Let dry. | #Let dry. | ||
| Line 45: | Line 40: | ||
#Immediately put on a thick slide. Then seal with nail polish so PBS+β-mercaptoethanol does not evaporate. | #Immediately put on a thick slide. Then seal with nail polish so PBS+β-mercaptoethanol does not evaporate. | ||
#Let dry. Do not expose to light. Ready to be imaged. | #Let dry. Do not expose to light. Ready to be imaged. | ||
[[UA Biophysics:Protocols|Return to Protocols]]<br> | |||
Latest revision as of 13:40, 18 July 2018
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
- Biotin-labeled tubulin (Cytoskeleton Inc., Denver, CO)
- Taxol (Sigma, Saint Louis MO)
- GTP [100mM]
- BRB80 buffer
- MgCl2 [50mM]
- DMSO
- AlexaFluor 488
- PBS-BSA 0.1%
- β-mercaptoethanol [14M]
- Wet chamber
- Coverslips and slides
- Nail polish
RECIPE
- Prepare 1 ml PBS+β-mercaptoethanol 14mM. Put aside.
- Prepare 13.0 μl of growth solution by adding in an eppendorf
- MgCl2 [50mM]: 1.04 μl
- GTP [25mM]: 2.00 μl
- DMSO [100%]: 0.65 μl
- BRB80: 9.40 μl
- From this growth solution extract 6.5 μl and pour 20μg of biotin-labeled tubulin (Cytoskeleton Inc., Denver, CO) in it. Incubate for 30min at 37°C. Discard the other 6.5μl .
- Prepare 500μl BRB80 + Taxol 10μM.
- Disolve the 6.5 μl of growth solution + polymerized microtubules in the 500μl BRB80 + Taxol 10μM.
- From the 506.5μl take 20μl and deposit it on a thin slide.
- Let dry.
- Rinse with PBS-BSA 0.1% and leave for 30min at room temperature.
- Add 20μl of Alexa 488 2000x in PBS.
- Leave in a humid petri dish at room temperature for 30min.
- Put a droplet of PBS+β-mercaptoethanol (~20μl).
- Immediately put on a thick slide. Then seal with nail polish so PBS+β-mercaptoethanol does not evaporate.
- Let dry. Do not expose to light. Ready to be imaged.
