F/G-actin ratio

F/G Actin separation protocol 

It is important that ALL buffers are made fresh and are ice-cold prior to use.

Lysis buffer recipe (for G-actin supernatant)

10mM K₂PO₄

100mM NaF

50mM KCl

2mM MgCl₂

1mM EGTA

0.2mM DTT

0.5% Triton-X 100

1mM sucrose 

pH 7.0

Second lysis buffer (for F-actin supernatant)

1.5mM guanidine hydrochloride

1mM sodium acetate

1mM CaCl₂

1mM ATP

20mM Tris-HCl

pH 7.5

Protocol

1. Prepare both lysis buffers in diH₂O and place on ice. 
2. Lyse cells or homogenates with sufficient volume of G-actin supernatant lysis buffer and centrifuge at 15,000g for 30 minutes
3. Transfer the supernatant (this is your G-actin fraction) into a fresh tube and place aside
4. Resuspend the pellet from the initial centrifugation in lysis buffer (G-actin buffer) plus equal volume of second lysis buffer (F-actin buffer) and incubate on ice for one hour with gentle mixing every 15 mins to convert F-actin into soluble G-actin. 
5. Centrifuge samples at 15,000g for 30 min and transfer supernatant (F-actin) to a fresh tube
6. If possible, run samples that same day on an immunoblot (load equal volumes)