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MOLECULAR BIOLOGY: WORKING WITH DNA

PROTEIN EXPRESSION: LABELING

Preparation of Rhodamine-Labeled Actin

Preparation of Rhodamine-Labeled Actin
Contributor: The Laboratory of David Drubin at the University of California, Berkeley
 
Procedure
1. Polymerize rabbit muscle actin (10 mg) in Polymerization Buffer for 2 hours at 4°C. Layer onto a cushion of Polymerization Buffer with 40% (v/v) glycerol. Centrifuge in a TLA-100.3 rotor at 80,000 rpm (265,000 X g) for 1 hr.

2. Aspirate the cushion and add 1 to 2 pellet volumes of the Polymerization Buffer and resuspend the pellet by pipetting until it is a viscous slurry (see Hint #2).

3. Add NHS-rhodamine, dissolved in DMF (CAUTION! see Hint #1), to the actin slurry at 1 to 2 moles of the fluorochrome per mole of actin. Mix gently and incubate at room temperature in the dark for 40 min with gentle mixing (see Hint #3).

4. Stop the labeling reaction by adding 1.5 volumes of Stop Buffer and incubate at room temperature for 15 min.

5. Layer the actin on a cushion of 40% Glycerol (v/v) in F-Actin Buffer and centrifuge in a TLA-100.3 rotor at 80,000 rpm (265,000 X g) for 1 hr.

6. Resuspend the actin pellet in cold G-Buffer. Allow the pellet to swell for 30 min on ice, then resuspend the pellet by pipetting up and down (avoid making bubbles in the solution). Sonicate twice using a polytron homogenizer at an output level of 2 for 2 sec each.

7. Transfer the solution to a dialysis bag and dialyze against a large volume if G-Buffer for at least 20 hr at 4°C with at least 1 change of dialysis buffer.

8. Collect the depolymerized actin and centrifuge for 15 min in a TLA-100.3 rotor at 40,000 rpm (66,000 X g) at 4°C.

9. Run the actin through a G25 column that is 10- to15-fold the sample volume and equilibrated in G-Buffer. Collect the biggest (first) pink peak.

10. Polymerize the actin by adding KMA Buffer and incubate for 2 hr at 4°C in the dark.

11. Collect the sample and layer it on a cushion of polymerization Buffer with 40% (v/v) glycerol. Centrifuge in a TLA100-3 rotor at 80,000 rpm (265,000 X g) for 40 min at 4°C.

12. Aspirate the cushion and add an equal pellet volume of G-Buffer.

13. Swell the pellet on ice for 1 hr and then gently resuspend (avoiding bubbles) the pellet.

14. Sonicate twice using a polytron homogenizer at an output level of 2 for 2 sec each.

15. Transfer the solution to a dialysis bag and dialyze against a large volume if G-Buffer for at least 48 hr at 4°C with at least 3 changes of dialysis buffer.

16. Aliquot the rhodamine-labeled actin and freeze the aliquots in Liquid Nitrogen.

Solutions
Stop Buffer   0.1% 2-Mercaptoethanol
50 mM Tris-HCl, pH 8.0
100 mM Potassium Glutamate
1 mM ATP
5 mM MgCl2
Polymerization Buffer   1 mM ATP
5 mM MgCl2
100 mM HEPES, pH 8.6
KMA Buffer   100 mM KCl
1 mM ATP
5 mM MgCl2
G-Buffer (for dialysis)   0.2 mM ATP
0.2 mM DTT
5 mM Tris, pH 8.0
0.2 mM CaCl2
0.02% (w/v) Sodium Azide (CAUTION! see Hint #1)
G-Buffer   0.2 mM ATP
0.2 mM DTT
5 mM Tris, pH 8.0
0.2 mM CaCl2
F-Actin Buffer   0.1% 2-Mercaptoethanol
50 mM Tris-HCl, pH 8.0
1 mM ATP
5 mM MgCl2
50 mM KCl
 
BioReagents and Chemicals
Calcium Chloride
HEPES
DMF
Nitrogen, Liquid
ATP
2-Mercaptoethanol
Magnesium Chloride
Sodium Azide
DTT
Rhodamine
Potassium Glutamate
Tris-HCl
Potassium Chloride
 
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. The actin concentration should be 50 to 100 mg/ml.

3. From this step on, try to keep the actin in the dark.