Actin Assembly Timepoint Assay
This protocol is designed to test how well actin can assemble in various strains.
Preparation:
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50ml falcon for thawing cells (2 per strain)
1.5ml eppendorf tubes for low speed spin (4-5 per strain x 2)
15ml falcon for collecting LSS (1 per strain)
15ml falcon for collecting HSS (1 per strain)
15ml screw cap falcon for combining all initiation ingredients (1 per strain)
Timepoint pellet epps (1 pellet epp per timepoint per strain)
Boiling tubes for each pellet sample
TLA 100.3 tubes. (2 for HSS per strain, 1 0 timepoint to get started)
Also obtain 2 ice buckets.
Method:
RUN IN DUPLICATE!
Start with the following amount, and repeat for each strain:
For 2x concentration:
 5.0g cells
 2.5ml 2X HE
 1.5µl 1000X pepstatin
 1.5µl 1000X aqueous PI
 20µl 0.2M PMSF
Combine all ingredients in a 50ml falcon. When cells are completely thawed place into
4-5 epps/strain.
Spin: 3’ in microfuge in cold room. Remove LSS using a p1000 and place in falcon
tubes. Repeat for remaining samples that did not fit in first spin.
Place each LSS into 2 TLA 100.3 tubes
Spin (up to 3 strains) 15’, 80K, 4C, TLA 100.3
Remove HSS with p1000 and place into 15ml falcon. Be careful to avoid top lipid layer
and lower pellet.
Repeat for remaining samples.
Perform the colorimatric assay to determine the concentration of each sample and dilute
all samples to 10mg/ml:
 Make a 1:10 dilution of HSS (10 ul HSS in 90 ul HE)
 Make 1x buffer for assay (2ml 5x + 8 ml water)
 Use 10µl of 1:10 dilution in 1ml 1X SDS buffer
 Take OD590
 Use the following formula to adjust concentration:
(0.333) / (OD590) = X
(2 ml) x (X) = amount of HSS to use
(2 ml) – 2X = amount of buffer required to dilute HSS
Remove 500µl for “0” timepoint
Place the following into a 15ml falcon:
HSS
Glycerol
1M MgCl2
0.1M ATP
2.5M KCl
2.0ml
222µl
11µl
22µl
44µl
Let assembly happen in falcon tube and remove 500µl sample for spinning immediately
prior to the desired timepoint.
After all ingredients added, mix well, start timer, and spin “0” timepoint.
Spin 6’, 80K, 4C, TLA 100.3
Resuspend pellets in 50µl HE
For samples:
Place 30µl in 15µl of boiling 3X SDS buffer.