Product Uses Include
- Microinjection into muscle cell followed by electron microscopy of streptavidin conjugated gold particles to determine the cellular localization of the modified actin.
- In vitro mot ility experiments using biotinylated seeds of F-actin bound to fluorescent beads for laser tweezer manipulation.
- Produce affinity columns using biotinylated F-actin or G-actin to extract and purify novel proteins.
- Purify proteins or develop bioassays using biotin F-actin or G-actin with streptavidin-magnetic beads.
Material
Rabbit skeletal muscle actin (Cat. # AKL99) has been modified to contain covalently linked biotin at random surface lysine residues. An activated ester of biotin is used to label the protein. The labeling stoichiometry has been determined to be approximately 1 biotin per actin monomer. Biotinylated actin has an approximate molecular weight of 43 kDa. AB07 is supplied as a lyophilized powder. The lyophilized protein when stored desiccated to < 10% humidity at 4°C is stable for 6 months. Th e protein should be reconstituted to 10 mg/ml with distilled water. The protein will then be in the following buffer: 5 mM Tris-HCl pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 5% sucrose, and 1% dextran.
Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% polyacrylamide gel. AB07 biotinylated actin is >99% pure. No free biotin is apparent in the final product.
Biological Activity
The biological activity of biotinylated actin is determined from its ability to efficiently polymerize into filaments in vitro and separate from unpolymerized components in a spin down assay. Stringent quality control ensures that 80-90% of the biotinylated actin can polymerized in this assay. This is comparable to the polymerization capacity of unmodified actin (Cat. # AKL99).
To determine the efficiency of biotin labeling, nanogram amounts of biotinylated actin are separated by electrophoresis and electroblotted onto a nitrocellulose membrane. The blot is then probed with streptavidin linked alkaline phosphatase. Quality control ensures that the biotin label on actin can be detected down to the 1 ng level of protein. No free label is apparent in the final product.
