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DATASHEET: PURE ACTIN (>90%)

Catalog Number: 190101
Source: Rabbit Skeletal Muscle
Store Desiccated
Background:

Actin, a highly conserved cytoskeletal protein, is required for several essential eukaryotic processes including cell migration, muscle contraction, and cytokinesis. Actin (43 kDa) exists either as a free monomer termed G-actin (globular) or a linear polymer termed F-actin (filamentous). Given the asymmetry of G-actin, F-actin has inherent polarity with distinct “pointed” and “barbed” ends. Another critical feature of F-actin is its ability to treadmill, wherein G-actin is exchanged from both ends of the filament. This property emerges from the ATPase activity of G-actin and confers force-generating capabilities to F-actin. Mutations within actin and actin-associated proteins correlate with a variety of human diseases, including deafness and skeletal and cardiac myopathies.

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 α-actinin (100 kDa) and Cap Z (33 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.

Storage and Handling:

Store Pure Actin in a cool, dry environment. The product is stable under these conditions for 2 years. Reconstitute Pure Actin by resuspending in ice-cold ultrapure water to 9 mg/ml (note that 1 mg actin = 3 mg powder). Reconstituted Pure Actin can be maintained at 4°C for several weeks with the addition of 0.05% sodium azide. Alternatively, reconstituted Pure Actin can be aliquoted into experimental-sized batches, flash frozen in liquid Nitrogen, and stored at -80°C. Avoid repeated freeze-thaw cycles. When necessary, dilute reconstituted Pure Actin with Actin Working Buffer (Cat. No. 000102, 5 mM Tris-HCl and 0.2 mM CaCl2, pH 8.0) supplemented with 0.2 mM ATP and 0.5 mM DTT.

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.

Polymerization Protocol:

Dilute Pure Actin to 0.4 mg/ml with Actin Working Buffer (Cat. No. 000102, 5 mM Tris-HCl and 0.2 mM CaCl2, pH 8.0) supplemented with 0.2 mM ATP and 0.5 mM DTT. Incubate on ice for 1 hour followed by centrifugation at 14k rpm for 15 minutes at 4°C. Collect the supernatant and add 1/10 volume of Actin Polymer Buffer (10X; Cat. No. 000103; 500 mM KCl and 20 mM MgCl2). Incubate at room temperature for 1 hour to polymerize. The resulting actin filaments can be stored at 4°C for several weeks in the presence of 0.05% sodium azide.

Technical Notes:
  • store in a cool, dry environment
  • maintain in the presence of ATP and DTT
  • store at 4°C with 0.05% sodium azide upon reconstitution
  • regard actin concentration and KCl and MgCl2 addition when polymerizing

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.




pure-actin-purity

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.

pure-actin-polymerization

References:

1. Dráberová, E., Sulimenko, V., Sulimenko, T., Böhm, K., & Dráber, P. Recovery of tubulin functions after freeze-drying in the presence of trehalose. Analytical Biochemistry. 397, 67–72 (2010).

2. Spudich, J.A. & Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J. Biol. Chem. 246, 4866-4871 (1971).


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