Actin Thin Filaments (Rabbit skeletal)

On Arrival: 4°C
Briefly centrifuge to collect the product at the bottom of the tube. Reconstituting a tube of CS-MYS05 with 180 ul of Milli-Q water will generate a 1.4 mg/ml stock of gizzard S1 myosin in the following buffer: 8 mM PIPES pH 7.0, 8 mM MgCl2, 5% (w/v) sucrose and1% (w/v) dextran. The protein should not be exposed to repeated freeze-thaw cycles. The lyophilized protein is stable at 4C desiccated (<10% humidity) for 1 year.

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The cardiac thin filament complex (CTFC) has been assembled from purified F-actin (Cat. # AD99) and Tropomyosin / Troponin protein (TT) complex (Cat. # TT05). Thus, CTFC is composed of six proteins: Actin : Tropomyosin ??: Tropomyosin ? : Troponin C :Troponin I : Troponin T in a stoichiometric ratio of 7:1:1:1:1:1; see Figure 1. After assembly, the thin filaments are centrifuged at 100, 000 x g and the pellets resuspended in reaction buffer which purifies the calcium sensitive complex. The complex has been determined to be biologically active in a calcium activated myosin ATPase assay (see biological activity assay). The complex is supplied as a white lyophilized powder.

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 ul)
5. PM12 Reaction buffer (12 mM Pipes-NaOH, pH 6.8, 2 mM MgCl2).

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