|Tel :||01904 431402||Bioquote Limited|
The Raylor Centre
York, YO10 3DW
|Fax :||01904 431409|
|Int Tel :||00 44 1904 431402|
|Int Fax :||00 44 1904 431409|
Manufacturer: Cytoskeleton Inc
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 α: Tropomyosin β : Troponin C :Troponin I : Troponin T in a stoichiometric ratio of 7:1:1:1:1:1; 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.
Briefly centrifuge to collect the product to the bottom of the tube.The protein should be reconstituted to 2 mg/ml by the addition of room temperature (RT) PM12 buffer. Do not pipette vigorously as this will denature the filaments. A white solution will appear first. Leave this for 10 min, then centrifuge at 500 x g to remove air bubbles. The solution should now look clear. Do not centrifuge at high speed because the filaments will sediment. The protein will be in the following buffer: 32 mM PIPES pH 6.8, 25 mM KCl, 2 mM MgCl2, 5% (w/v) sucrose, and 1% (w/v) dextran.The protein solution can be frozen by aliquoting into "experiment sized" amounts, snap frozen in liquid nitrogen and stored at -70°C. The protein is stable for 6 months if stored at -70°C. The protein should not be exposed to repeated freeze-thaw cycles.The lyophilized protein is stable at 4°C desiccated (<10% humidity) for 1 year.
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% gradient polyacrylamide gel. The purity of the CTFC is >95%
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