Product Uses Include
Bovine cardiac muscle actin is supplied as a lyophilized powder. When one vial is reconstituted in 1 ml of Actin Monomer Buffer: 5.0 mM Tris-HCI pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP and 0.5 mM DTT, it is at a final concentration of 1.0 mg/ml.
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are > 99% pure.
The biological activity is determined in three ways. The first method utilizes pyrene muscle actin as a fluorescent indicator of polymerization (use a 1:10 ratio of AP07 to AD99) . The second involves sedimentation of actin polymer by high speed centrifugation. The third method measures the integrity of the monomer by the specific inhibition of DNase activity. The biological activity is > 90%.
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Question 1: What is the isotype composition of the cardiac actin from bovine heart muscle?
Answer 1: Bovine cardiac actin (Cat. # AD99) is composed of 84% a-cardiac and 16% a-skeletal actin isoforms.
Question 2: How do I measure actin polymerization with the pyrene assay when there is no available pyrene conjugate for cardiac actin?
Answer 2: To examine the polymerization of unlabeled cardiac actin (Cat. # AD99), please click here for a polymerization protocol that uses an excess of the unlabeled [cardiac] actin plus 10% of pyrene-labeled skeletal muscle actin. The pyrene muscle actin will not polymerize on its own at the concentration used in this assay, so the reaction is dependent on unlabeled cardiac actin (Cat. # AD99) for F-actin formation. In this way, the pyrene-labeled muscle actin is taken up and polymerized to serve as a reporter for polymerization of the unlabeled actin that is present at a much greater concentration.
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