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MOLECULAR BIOLOGY: WORKING WITH DNA

PROTEIN EXPRESSION: LABELING

Labeling Muscle Actin with Carboxyfluorescein Succinimidyl Ester

Labeling Muscle Actin with Carboxyfluorescein Succinimidyl Ester
Contributor: The Laboratory of Yu-Li Wang at the University of Massachusetts Medical School.
URL: Lab Web Site
 
Procedure
A. Day One Perform under reduced light and at 4°C unless otherwise noted.

1. Resuspend 10 mg lyophilized Actin in 2 ml Buffer 1. Be careful not to make bubbles.

2. Add DTT to 0.5 mM final concentration.

3. Dialyze against 250 ml Buffer 1 for 8 hr (or overnight).

4. Prepare according to manufacturers instructions washed G-25 column (30 x 1.5 cm) and equilibrate G-25 column with Buffer 5.

5. Collect Actin from dialysis tubing and transfer to a small vial with a stir bar.

6 Measure the volume and add KCl to 70 mM (final concentration) and CaCl2 to 1 mM (final concentration).

7. Incubate at room temperature for 30 min.

8. Add 166 μl (8.31 mg) CFSE Stock to 1 ml of Buffer 2 while vortexing intermittently after adding a every couple of drops.

9. Mix dye solution immediately with Actin solution, by gentle pipetting or stirring.

10. Incubate at room temperature for 1 hr.

11. Clarify solution by centrifugation at 106,000 X g for 1 hr at 4°C (40,000 rpm using a Type 50Ti rotor).

12. Discard the supernatant and resuspend the pellet in 1.0 ml of Buffer 3.

13. Measure the total volume.

14. Slowly add an equal volume of Buffer 4 while stirring.

15. Stir gently on ice for 30 min.

16. Clarify solution by centrifugation at 39,000 X g for 20 hr at 4°C (18,000 rpm using a SS34 rotor rotor).

17. Immediately remove the supernatant and pass through the equilibrated G-25 column

18. Collect 10 drop fractions.

19. Collect fluorescent fractions in the void volume and measure the volume in a volumetric conical tube.

20. Polymerize Actin by adding KCl to 100 mM (final concentration) and MgCl2 to 2 mM (final concentration).

21. Incubate for 30 to 60 min at room temperature.

22. Centrifuge at 106,000 X g for 1 hr at 15°C (40,000 rpm using a Type 50Ti rotor).

23. Soak pellet in 0.6 ml Buffer 5 for 1 to 2 hr.

24. Resuspend by gentle pipetting.

25. Dialyze resuspended Actin against Buffer 5 overnight at 4°C.

B. Day Two

1. Remove the Actin from dialysis tubing.

2. Centrifuge Actin at 106,000 X g for 1 hr at 4°C (40,000 rpm using a Type 50Ti rotor).

3. Polymerize Actin by adding KCl to 100 mM (final concentration) and MgCl2 to 2 mM (final concentration).

4. Incubate for 30 to 60 min at room temperature.

5. Centrifuge Actin at 106,000 X g for 1 hr at 4°C (40,000 rpm using a Type 50Ti rotor).

6. Soak pellet in 0.4 ml Buffer 5 for 1 to 2 hr

7. Resuspend by gentle pipetting.

8. Dialyze resuspended Actin against Buffer 5 overnight.

9. Centrifuge Actin at 106,000 X g for 1 hr at 4°C (40,000 rpm using a Type 50Ti rotor).

10. Resuspend Actin in Buffer 5.

11. Measure concentration and dye/protein molar ratio by diluting 1:40 with the Buffer 5 and read the optical density at 495 nm.

D/P = {OD495 x 41 / 60,000} / {(mg/ml) / 43,000}

Where:

OD495 is the optical density at 495 nm

mg/ml is the Actin protein concentration

12. Dilute sample to 3 to 5 mg/ml with the Buffer 5.

13. Calculate total mg of Actin and dissolve 2 mg Sucrose per mg Actin.

14. Drop freeze aliquots in liquid nitrogen.

15. Dialyze against buffer 6 overnight before microinjection.

Solutions
10 mM CaCl2
0.7 M KCl
CFSE Stock   50 mg/ml Carboxyfluorescein Succinimidyl Ester
Prepared in DMSO
Buffer 6   0.2 mM ATP
2 mM Tris-Acetate, pH 6.95
0.05 mM MgCl2
Buffer 5   2 mM Tris-HCl, pH 8.0
0.5 mM ATP
0.2 mM CaCl2
0.5 mM DTT
pH and store at 4°C
Buffer 4   50 mM Tris-HCl, pH 8.0
1.2 M KI
3 mM CaCl2
Buffer 3   5 mM DTT
10 mM Glutamic Acid
16 mM ATP
20 mM Tris-HCl, pH 8.0
5 mM Lysine
0.2 mM CaCl2
Buffer 2   150 mM PIPES, pH 7.0
70 mM KCl
0.5 mM ATP
0.2 mM CaCl2
Buffer 1   1 mM PIPES, pH 6.8
0.5 mM ATP
0.2 mM CaCl2
pH and store at 4°C
 
BioReagents and Chemicals
ATP
Liquid nitrogen
Sucrose
DMSO
Potassium Chloride
Tris-Acetate
Potassium Iodide
Glutamic Acid
Lysine
Magnesium Chloride
Ice
Calcium Chloride
Tris-HCl
DTT
Carboxyfluorescein Succinimidyl Ester
PIPES
 
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