Actin Assembly Timepoint Assay This protocol is designed to test how well actin can assemble in various strains. Preparation: 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, 4C, 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, 4C, TLA 100.3 Resuspend pellets in 50µl HE For samples: Place 30µl in 15µl of boiling 3X SDS buffer.