PURE ACTIN (>90%)

Catalog Number: 190101
Source: Rabbit Skeletal Muscle

Material

Pure Actin is extracted from rabbit skeletal muscle using an optimized version of the method of Spudich and Watt (1971) and lyophilized by an adaptation of the method of Dráberová et al. (2010). The resulting product is >90% pure (Figure 1) and >80% polymerization competent (Figure 2). Possible contaminants include a-actinin (100 kDa, Figure 1). For actin >99% pure, see our Ultra-Pure Actin product (Cat. No. 160101). Pure Actin is supplied as a white powder with an actin-to-powder weight ratio of 1:3. When reconstituted with ultrapure water to 9 mg/ml, the buffer conditions are 2 mM Tris-HCl, 0.2 mM CaCl2, 0.2 mM ATP, 1 mM DTT, and 0.25 M Trehalose, pH 8.0.

Activity

When supplemented with KCl and MgCl2, Pure Actin will polymerize into filaments when above its critical concentration. The recommended actin concentration for ensuring polymerization is 0.4 mg/ml.

Uses

Pure Actin is supplied for use in cell-free experimental systems including structural, biochemical, and biophysical studies.

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Figure 1: Pure Actin is >90% pure. Coomassie G250-stained protein gel of Pure Actin separated by SDS-PAGE. The actin appears as the majority species migrating at ~43 kDa. Possible contaminants include a-actinin (100 kDa) and are not in excess of 10%. Molecular weight markers (kDa) and loaded protein quantities are indicated.




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Figure 2: Pure Actin is >80% polymerization-competent. Pure Actin was polymerized in the absence (-PB) or presence (+PB) of Actin Polymer Buffer (10X, Cat. No. 000103; 50 mM KCl and 2 mM MgCl2) followed by centrifugation at 48k rpm for 1 hour. Pellet (P) and supernatant (S) fractions were collected and subjected to SDS-PAGE and Coomassie G250-staining. >80% of Pure Actin was incorporated into filaments as determined by measuring the residual protein concentration in the supernatant fraction.

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