Actin-Toolkit Fluorometry with Pyrene Actin
Pyrene-labeled native α-actin from rabbit skeletal muscle
Cat. #: 8030-01
Applications
- Kinetic analysis of effects of purified ligands, cell extracts or tissue extracts on actin polymerization and depolymerization.
- Analysis of the influence of divalent cations or pharmacological compounds on actin assembly and disassembly.
- Measurement of F-actin severing or depolymerizing activity under steady-state conditions.
Description
| Product name | Actin-Toolkit Fluorometry with Pyrene Actin |
| Protein | Pyrene Actin (α-actin) |
| Origin | Rabbit skeletal muscle |
| Molecular mass | 42 kDa |
| Toolkit description |
The Actin-Toolkit Fluorometry is designed for kinetic analysis of actin polymerization and depolymerization using pyrene-labeled actin as a fluorescence reporter. Pyrene fluorescence increases strongly upon incorporation of labeled actin into F-actin, allowing continuous monitoring of actin assembly and disassembly. The assay can be used to examine the effects of purified proteins, recombinant fragments, lipids, pharmacological compounds, cell extracts or tissue extracts on actin dynamics under defined in vitro conditions. The kit contains 4 × 1.0 mg actin with 10% pyrene labeling. Pyrene is introduced at Cys374 using N-(1-pyrene)iodoacetamide. Gelsolin, actin buffer components, PolyMix and CaCl2 are supplied to support assay setup and controlled variation of polymerization conditions. A dedicated handbook describes fluorometric actin-polymerization and depolymerization assays. For experiments requiring a higher pyrene fraction, 100% Pyrene Actin may be used as an additional reagent. |
| Labeling | 10% pyrene-labeled actin; labeling at Cys374 with N-(1-pyrene)iodoacetamide. |
| Purity | >99% by scanning densitometry. |
Properties
| Form | Lyophilized. |
| Kit content | 4 × 1.0 mg Pyrene Actin 10% (lyophilized powder); 1 × 20 µg Gelsolin; 1 × 1000 ml ATP-Actin Buffer, supplied as components; 1 × 1.0 ml PolyMix (10× stock); 1 × 0.5 ml CaCl2 (100 mM); 1 × Toolkit Handbook Fluorometry; 10 × black tubes and tags. |
| Actin buffer after reconstitution | 2 mM Tris-Cl, pH 8.2, 0.4 mM ATP, 0.5 mM DTT, 0.1 mM CaCl2 and 0.3% disaccharides when reconstituted with 1.0 ml ultrapure water to obtain a 1 mg/ml actin solution. |
| Related products | Actin-binding proteins and 100% Pyrene Actin for experiments requiring an increased pyrene-labeled fraction. |
| Purification notes | Native actin purified from rabbit skeletal muscle; final purification by gel permeation chromatography (GPC). Pyrene conjugation is performed on purified actin. |
| Protein concentration | Actin concentration is determined spectrophotometrically from A290 after correction for the absorbance contribution of the pyrene label. For unlabeled actin, A290nm 1% = 0.62. |
| Storage instructions | We guarantee stable performance of the kit components for 6 months when stored at −70 °C upon arrival. Solubilized G-actin should be kept on ice and used within 1 week. Avoid refreezing. |
| Shipping conditions | Shipped at ambient temperature. Upon delivery store at −70 °C. |
| CAS no. | 51005-14-2 (Actin) |
| Remarks | For research use only. Not for diagnostic, therapeutic, clinical or in vivo use. |
Buffers and proteins from Hypermol® are made using ultrapure reagents in Milli-Q™ water, as described in our publications.
Further Information
Handbook Actin-Toolkit Fluorometry
(Web version only for demonstration purpose!)
A full print version with protocols come with the kit.
Flyer Actin-Toolkits
Material and Safety Data Sheet
Protein Sequence on NCBI
