Cat. # AB07

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 motility 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. The 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. (see Figure 1.). No free biotin is apparent in the final product.

Figure 1. AB07 purity determination. A 10 µg sample of biotinylated actin (molecular weight approx. 43 kDa) was separated by electrophoresis in a 12% SDS-PAGE system. The protein was stained with Coomassie Blue. Protein quantitation was determined with the Precision Red Protein Assay Reagent (Cat.# ADV02).

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 (see Figure 2). No free label is apparent in the final product.

Figure 2. Detection of 1 ng of biotinylated actin. Serial dilutions of biotinylated actin were separated by electrophoresis on a 12% polyacrylamide gel and blotted onto Nitrocellulose. The membrane was then probed with streptavidin alkaline phosphatase (Sigma) and detected with the 1-Step NBT/BCIP reagent (Pierce). Lane 1: 1000 ng, Lane 2: 100 ng, Lane 3: 10 ng, and Lane 4: 1 ng of biotinylated actin.