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Actin Thin Filaments (Bovine cardiac)

Actin Thin Filaments (Bovine cardiac)

Item no.	TFC01
Manufacturer	Cytoskeleton
Amount	1 x 1 mg
Category	Peptides & Proteins Recombinant Proteins Cell Biology Cytoskeleton Microfilaments
Type	Proteins
Specific against	other
Purity	Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% gradient polyacrylamide gel. The myosin and its light chains used to produce the myosin S1 fragment was determined to be 90% pure (see Figure 2).
Citations	1. J.M Murray. 1982. Hybridization and reconstitution of the thin filament. Methods Enzymol. 85: 15-17. 2. M.J. Holroyde et al. 1980. The calcium and magnesium binding sites on cardiac troponin their role in the regulation of myofibrillar adenosine triphosphatase. J. Biol. Chem. 255: 11688-11693.
ECLASS 10.1	32160409
ECLASS 11.0	32160409
UNSPSC	12352202
Shipping Condition	Room temperature
Available
Shipping Temperature	AT
Storage Conditions	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.
Description	* Limited stock available. If stock is not available, Cytoskeleton will produce a new batch upon request. Minimum order will apply. Inquire for more information.
Weight (grams)	9
Product Uses	Measurement of calcium activated myosin ATPase activityWhen bound to thin filaments. Identification/characterization of proteins or small moleculesthat affect the CTFC regulation and myosin ATPase activity Identification/characterization of proteins or small molecules that affect myosin / F-actin interaction
Material	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.
Biological Activity	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 uM 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 ul) 5. PM12 Reaction buffer (12 mM Pipes-NaOH, pH 6.8, 2 mM MgCl2).
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 ?l of PM12 800 ?l 5x MSEG (this is a BK051 component) 500 ?l of TFC01 30 ?l of Myosin S1 solution 20 ?l of 100 mM ATP 40 ?l of 100x PNP (this is a BK051 component) 3. Using the pre-warmed half area 96-well plate, pipette the following: 4. Pipette 10 ?l of 100 ?M calcium chloride into "activated" wells. 5. Pipette 10 ?l of Milli-Q water into "non-activated" wells. 6. Pipette 10 ?l of 10X test compound into appropriate wells. 7. Incubate at 37C for 2 min to warm the mixture. 8. Pipette 100 ?l of Myosin/Thin Filament mixture into all wells. 9. Start protocol, 41 readings, 30 seconds apart, 37C , OD 360+/- 5 nm. 10. Calculate Vmax and compare non-activated to calcium activated samples.
Equipment needed	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
Figure 1 Legend	Legend: Myosin is a hexameric protein consisting of two heavy chains and two light chains. Myosin can be proteolytically cleaved into heavy meromyosin (HMM) and light meromyosin (LMM) by ?-chymotrypsin in the presence of magnesium. In the presence of EDTA, however, ?-chymotrypsin produces the soluble myosin S1 fragment (3).

Note: The presented information and documents (Manual, Product Datasheet, Safety Datasheet and Certificate of Analysis) correspond to our latest update and should serve for orientational purpose only. We do not guarantee the topicality. We would kindly ask you to make a request for specific requirements, if necessary.

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TFC01-datasheet.pdf

TFC01-safety-datasheet.pdf