Actin Pelleting Assay

Overview


At relatively high centripetal forces, filamentous F-actin will pellet while monomeric G-actin will remain suspended in solution. This biophysical property can be exploited in probing for actin-binding agents and assessing actin polymerization competency.

For general actin handling instructions, please see “Storing and Handling Ultra-Pure Actin” or “Storing and Handling Pure Actin”.

Overview


At relatively high centripetal forces, filamentous F-actin will pellet while monomeric G-actin will remain suspended in solution. This biophysical property can be exploited in probing for actin-binding agents and assessing actin polymerization competency.

For general actin handling instructions, please see “Storing and Handling Ultra-Pure Actin” or “Storing and Handling Pure Actin”.

Recommended Products


Unlabeled Tubulin

Unlabeled Tubulin

Buffers

Recommended Products


Recommended Products

Unlabeled Tubulin

Buffers

Overview


At relatively high centripetal forces, filamentous F-actin will pellet while monomeric G-actin will remain suspended in solution. This biophysical property can be exploited in probing for actin-binding agents and assessing actin polymerization competency.

For general actin handling instructions, please see “Storing and Handling Ultra-Pure Actin” or “Storing and Handling Pure Actin”.

Recommended Products


Unlabeled Actin


Buffers


Protocol


1. Assemble

Assemble the polymerization reaction on ice by adding 1/10th volume Actin Polymer Buffer (10X) to actin at 0.4 mg/ml.

Add ATP to 1 mM.

2. Polymerize

Incubate the polymerization reaction at room temperature for 1 hour.

3. Pellet

Spin actin solutions at 48,000 rpm for 1 hour at 26°C in an ultracentrifuge.

4. Collect Fractions


  • Collect the supernatant.
  • Add an equal volume of 2X Sample Buffer to the supernatant fraction.
  • Gently rinse the pellet twice with Actin Working Buffer.
  • Resuspend the pellet in 1X Sample Buffer to a final volume matching that of the supernatant fraction.

5.Analyze

Analyze fractions by SDS-PAGE.

Protocol


Step 1

1. Assemble

Assemble the polymerization reaction on ice by adding 1/10th volume Actin Polymer Buffer (10X) to actin at 0.4 mg/ml.

Add ATP to 1 mM.

Step 2

2. Polymerize

Incubate the polymerization reaction at room temperature for 1 hour.

Step 3

3. Pellet

Spin actin solutions at 48,000 rpm for 1 hour at 26°C in an ultracentrifuge.

Step 4

4. Collect Fractions


  • Collect the supernatant.
  • Add an equal volume of 2X Sample Buffer to the supernatant fraction.
  • Gently rinse the pellet twice with Actin Working Buffer.
  • Resuspend the pellet in 1X Sample Buffer to a final volume matching that of the supernatant fraction.

Step 5

5.Analyze

Analyze fractions by SDS-PAGE.

Protocol


Step 1

1. Assemble

Assemble the polymerization reaction on ice by adding 1/10th volume Actin Polymer Buffer (10X) to actin at 0.4 mg/ml.

Add ATP to 1 mM.

Step 2

2. Polymerize

Incubate the polymerization reaction at room temperature for 1 hour.

Step 3

3. Pellet

Spin actin solutions at 48,000 rpm for 1 hour at 26°C in an ultracentrifuge.

Step 4

4. Collect Fractions


  • Collect the supernatant.
  • Add an equal volume of 2X Sample Buffer to the supernatant fraction.
  • Gently rinse the pellet twice with Actin Working Buffer.
  • Resuspend the pellet in 1X Sample Buffer to a final volume matching that of the supernatant fraction.

Step 5

5.Analyze

Analyze fractions by SDS-PAGE.