User:Nadiezda Fernandez-Oropeza/Notebook/Notebook/2011/05/18: Difference between revisions

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# First turn on the mercury lamp and prepare the microscope for Kohller Illumination.
# First turn on the mercury lamp and prepare the microscope for Kohller Illumination.
# Polymerize the microtubules (Procedure can be found here[User:Nadiezda_Fernandez-Oropeza/Notebook/Notebook/2011/05/16 Polimarization of microtubules]).
# Polymerize the microtubules (Procedure can be found here[[User:Nadiezda_Fernandez-Oropeza/Notebook/Notebook/2011/05/16 Polimarization of microtubules]]).
# In the 30 minute wait for the polymerization of the microtubules, one can prepare the following solutions:
# In the 30 minute wait for the polymerization of the microtubules, one can prepare the following solutions:
#* 0.5 mg/mL of α-Casein in PEM: 500 µL of 1mg/mL α–Casein in PEM and 500 mL of PEM.
#* 0.5 mg/mL of α-Casein in PEM: 500 µL of 1mg/mL α–Casein in PEM and 500 mL of PEM.
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#* Kinesin in PEM-αA: 2 µL of Kinesin with 18 µL of PEM-αA.
#* Kinesin in PEM-αA: 2 µL of Kinesin with 18 µL of PEM-αA.
# In a flow cell, let 10 µL of 1mg/mL α–Casein in PEM rest for 10 minutes.
# In a flow cell, let 10 µL of 1mg/mL α–Casein in PEM rest for 10 minutes.
# During this 10 minutes, prepare the Motility solution (Procedure can be found here [User:Nadiezda_Fernandez-Oropeza/Notebook/Notebook/2011/05/17#Motility_solution Motility Solution]).
# During this 10 minutes, prepare the Motility solution (Procedure can be found here [[User:Nadiezda_Fernandez-Oropeza/Notebook/Notebook/2011/05/17#Motility_solution Motility Solution]]).
# After the 10 minutes are up, exchange the fluid on the cell with the solution of Kinesin in PEM-αA, and let it incubate for 5 minutes.
# After the 10 minutes are up, exchange the fluid on the cell with the solution of Kinesin in PEM-αA, and let it incubate for 5 minutes.
# After those five minutes, exchange the fluid on the cell with 20 µL of the motility solution.
# After those five minutes, exchange the fluid on the cell with 20 µL of the motility solution.

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Gliding Motility Assay

  • This entry describes the procedure in the Gliding Motility Assay

Procedure

  1. First turn on the mercury lamp and prepare the microscope for Kohller Illumination.
  2. Polymerize the microtubules (Procedure can be found hereUser:Nadiezda_Fernandez-Oropeza/Notebook/Notebook/2011/05/16 Polimarization of microtubules).
  3. In the 30 minute wait for the polymerization of the microtubules, one can prepare the following solutions:
    • 0.5 mg/mL of α-Casein in PEM: 500 µL of 1mg/mL α–Casein in PEM and 500 mL of PEM.
    • PEM–αA: 1µL of PEM-A and 99 µL of 0.5 mg/mL of α-Casein in PEM.
    • Kinesin in PEM-αA: 2 µL of Kinesin with 18 µL of PEM-αA.
  4. In a flow cell, let 10 µL of 1mg/mL α–Casein in PEM rest for 10 minutes.
  5. During this 10 minutes, prepare the Motility solution (Procedure can be found here User:Nadiezda_Fernandez-Oropeza/Notebook/Notebook/2011/05/17#Motility_solution Motility Solution).
  6. After the 10 minutes are up, exchange the fluid on the cell with the solution of Kinesin in PEM-αA, and let it incubate for 5 minutes.
  7. After those five minutes, exchange the fluid on the cell with 20 µL of the motility solution.
  8. Finally, seal the end of the sample section on the flow cell with nail polish.



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