Product Uses Include
α-actinin has been purified from rabbit skeletal muscle. The protein is supplied as a lyophilized powder and upon reconstitution, will be in the following buffer: 4 mM Tris-HCl pH 7.6, 4 mM NaCl, 20 μM EDTA, 1% (w/v) sucrose, and 0.2% (w/v) dextran. The lyophilized product is stable at -70°C for at least 2 years. AT01 should be resuspended in nanopure water containing 1 mM β-mercaptoethanol or DTT to the desired working concentration.
Purity is determined by scanning densitometry of the protein on an SDS-PAGE gel. α-actinin is >90% pure.
Figure 1: α-actinin protein purity determination. 20 µg of AT01 was run on an SDS-PAGE gel and stained with coomassie blue.
At 2.5 µM (0.25 mg/ml) α-actinin and 25 µM (1.0 mg/ml) F-actin at pH 7.0 and 24°C, approximately 70% of the α-actinin will co-sediment with F-actin after centrifugation at 150,000 x g for 1 h.
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Question 1: What is the source of the alpha-actinin Cytoskeleton sells?
Answer 1: Cytoskeleton’s alpha-actinin (Cat. # AT01) is purified from rabbit skeletal muscle and has a purity of >85%.
Question 2: Can this alpha-actinin be used for actin bundling experiments?
Answer 2: Yes, Cytoskeleton’s alpha-actinin (Cat. # AT01) is well-suited for actin bundling experiments using skeletal muscle actin, it will not work with non-muscle isoforms of actin. Briefly, α-actinin is tested for biological activity by a co-sedimentation assay with Factin. At 2.5 μM skeletal muscle α-actinin and 25 μM actin in the form of F-actin, >70% α-actinin will co-sediment with F-actin. The reaction is carried out at pH 7.0 for 30 minutes at 24°C (at pH 8.0 α-actinin will not bind). Spin the mixture at 14,000 x g for 10 minutes. Probe a western blot with antibodies against alpha-actinin and actin. An SDS-PAGE gel is loaded with supernatant and pellet samples from alpha-actinin + actin and actin alone. After running the gel to separate samples it is stained with coomassie blue and then analyzed for band intensity at 43 and 116 kDal.
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