Legend: A 20 μg sample of CTFC proteins was separated by electrophoresis in a 4-20% SDS-PAGE system (Lane 2) and stained with Coomassie Blue. Molecular weights are 43 kDa actin,38 kDa Troponin T, 32 kDa Tropomyosin (α+β), 24 kDa Troponin I, and 18 kDa Troponin C. Protein concentration was measured with the Precision Red™ Protein Assay Reagent (Cat.# ADV02). See Blue molecular weight markers (Lane 1) are from Life Technologies Inc.

Biological Activity Assay

The biological activity of the CTFC can be determined from its ability to regulate myosin ATPase activity. The CTFC re-creates coated filaments which are analogous to the thin filaments of muscle fibers. Myosin is added in substoichiometric amounts and the reaction is initiated with ATP and calcium. Stringent quality control ensures that in the absence of exogenous calcium, the CTFC completely inhibits myosin ATPase. On addition of 10 µM calcium, myosin ATPase will be restored. Calcium binds to Troponin C which dissociates from F-actin, allowing myosin to bind.

Reagents

1. Cardiac Thin Filament Complex (1 x 1 mg, # TFC01)2. Cardiac Myosin S1 (0.25 mg, # MYS03)3. ATPase Assay Biochem Kit (Cat. # BK051)4. 100 mM ATP in 50 mM Tris-HCl pH 7.5 (100 µl)5. PM12 Reaction buffer (12 mM Pipes-NaOH, pH 6.8, 2 mM MgCl2).

Equipment

1. Spectrophotometer capable of measuring absorbance at 360 nm (+/- 5 nm bandwidth). We recommend a Spectra-Max M2 (Molecular Devices). Filter based machines are not suitable.2. Half area 96 well microtiter plate (Corning Cat.# 3696 or 3697)3. Multi-channel pipette

Method

The following major steps are recognized:

Step 1. Assemble required reagents and compounds (30 min).

Step 2. Prepare Thin Filament stock (15 min).

Step 3. Prepare Motor Mix and plate reader (15 min).

Step 4. Pipette Motor Mix into wells and start reaction/plate reader (10 min).

Thin Filament stock

1. Gently resuspend 1 x 1 mg TFC01 with room temp PM12 buffer to 2 mg/ml; it will be a white solution (500 ul per vial for 1 mg vial).2. Incubate at RT for 10 min.3. Centrifuge at 500 x g for 30s; now it is a clear solution.4. Store at room temperature for up to 20 min. 

Myosin reaction stock

1. Dilute S1 myosin to 1.0 mg/ml with ice cold PM12 buffer.2. Mix the following in the stated order at RT, to make 4.0 ml of Myosin/Thin Filament control mixture:

2610 μl of PM12

800 μl 5x MSEG (this is a BK051 component)

500 μl of TFC01

30 μl of Myosin S1 solution

20 μl of 100 mM ATP

40 μl of 100x PNP (this is a BK051 component)

3. Using the pre-warmed half area 96-well plate, pipette the following:4. Pipette 10 μl of 100 μM calcium chloride into “activated” wells.5. Pipette 10 μl of Milli-Q water into “non-activated” wells.6. Pipette 10 μl of 10X [test compound] into appropriate wells.7. Incubate at 37°C for 2 min to warm the mixture.8. Pipette 100 μl of Myosin/Thin Filament mixture into all wells.9. Start protocol, 41 readings, 30 seconds apart, 37°C , OD 360+/- 5 nm.10. Calculate Vmax and compare non-activated to calcium activated samples.

Figure 3: Calcium Dose Response Curve