Actin protein (biotin): skeletal muscle
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 (20 µg of protein) is supplied as a lyophilized powder.
Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% polyacrylamide gel. Biotinylated actin was found to be >99% pure. (see Figure 1.)
Applications
| Application | Reference |
Actin organization and its impact on ABP function and motion | 1, 2 |
| Modeling in vitro bio membranes | 3, 4 |
| Molecular Mechanisms underlying skeletal mediated force/stress | 5, 6 |
Actin and microtubule coupling, mechanical properties, and dynamics | 7, 8 |
Motor communication and function: motility assays, optical tweezers and optical traps | 9, 10, 11, 12 |
| Study actin binding proteins | 13, 14 |
| Applications in functional nanodevices | 15, 16 |
Figure 1: Biotinylated Actin Protein Purity Determination
Legend-Fig. 1: A 20 µg sample of biotinylated actin (Lanes 1 & 2) 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). Mark12 molecular weight markers are from Invitrogen.
Figure 2: Detection of biotinylated actin
Legend-Fig. 2: Serial dilutions of biotinylated actin were separated by electrophoresis on a 12% polyacrylamide gel and blotted onto PVDF. The membrane was then probed with streptavidin alkaline phosphatase and detected with the 1-Step NBT/BCIP reagent (Pierce). Lane 1. 100 ng, Lane 2. 10 ng, Lane 3. 1 ng of biotinylated actin.
Biological Activity Assay
The biological activity of biotinylated actin can be 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 >85% of the biotinylated actin can polymerized in this assay. This is comparable to the polymerization capacity of unmodified actin (Cat. # AKL99). The assay is carried out as described in the datasheet.
Application References
1- F-actin architecture determines consraints on myosin thick filament motion. 2022. Muresan C. et al. Nature Commun. 13, 7008
2 - α-catenin swithches between a slip and an asymmetric catch bond with F-actin to cooperatively regulate cell junction fluidity. Naure Commun. 13, 1146
3 - Design and construction of a multi-tiered minimal actin cortex for structural support in lipid bilayer applications. 2024. Smith A.J. et al. Appl. Bio. Mater. 7: 1936-1946
4 - Encapsulated actomyosin patterns drive cell-like membrane shape changes. 2022. Bashirzadeh Y. et al. iScience. 25 (5), 104236
5 - Reconstituting and characterizing actin-microtubule composites with tunable motor driven dynamics and mechanics. 2022. Sasanpour M. et al. Jove J. 10.3791/64228
6 - Molecular mechanism for direct actin force-sensing by alpha-catenin. 2020. Mei L. et al. eLife 9:e62514
7 - Visualizing Actin and Microtubule Coupling Dynamics In Vitro by TIRF Microscopy. 2022. Henty-Ridilla J. JoVE J. 10.3791/64074
8 - Actin and microtubule crosslinkers tune mobility and control co-localization in a composite cytoskeletal network. 2020. Farhadi L. et al. Soft Matter. 31.
9 - A binding protein regulated myosin-7a dimerization and actin bundle assembly. Liu R. et al. Nature Commun. 2021. 12, 563
10 - Myosin-specific adaptations of in vitro fluorescence microscopy-based motility assays. Tripathi A. et al. 2021. JoVE J. 10.3791/62180
11 - Probing myosin ensemble mechanics in actin filament bundles using optical tweezers. Al Azzam O. et al. 2022. JoVE J. 10.3791/63672
12 - High-speed optical traps address dynamics of processive and non-processive molecular motors. Gardini L. et al. 2022. Optical Tweezers. 2478.
13- Secreted gelsolin inhibits DNGR-1-dependent cross-presentation and cancer immunity. 2021. Cell 184: 4016-4031
14- Mitotic spindle positioning protein (MISP) preferentially binds to aged F-actin. 2024. Morales E.A. et al. J. Biol. Chem. 300(5) 107279
15- Comparison of actin-and microtubule-based motility systems for application in functional nanodevices. 2021. Reuther C. et al. New J. Phys. 23:075007
16- The potential of myosin and actin in nanobiotechnology. 2023. Mansson A. J. Cell Sci. 136: 10.1242/jcs.261025
For product Datasheets and MSDSs please click on the PDF links below. For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com
Question 1: Does biotinylated actin have the same polymerization dynamics as unlabeled actin?
Answer 1: The biological activity of biotinylated actin (Cat. # AB07) can be 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 polymerize in this assay. This is comparable to the polymerization capacity of unmodified actin (Cat. # AKL99).
Question 2: Can the biotinylated actin be used in a pull-down format to capture novel actin binding proteins?
Answer 2: Yes, the biotinylated actin (Cat. # AB07) can be used to pull-down actin binding proteins with streptavidin beads. Below is a protocol:
1. Polymerize biotin actin at 0.4 mg/ml, using 20 mg AB07 plus 180 mg of unlabeled actin (Cat. # AKL99) for 1 h at RT, (alternatively for a monomer binding test, use AB07 diluted to 0.4 mg/ml in A-buffer for 1 h at RT, and add that to the beads 1 mg per 1 ml of beads)
2. Stabilize with 1 mM phalloidin (Sigma) added from 200 mM stock in methanol, diluted to 20 mM in F-actin buffer.
3. Mix with 200 ml packed volume of streptavidin beads washed with F-actin + phalloidin buffer. Incubate for 1 h at RT on a rotator 20 rpm, then wash again with 2 x 1 ml of F-actin + phalloidin buffer.
4. Use 20 mg AB07 per reaction = 20 ml of beads, plus 1 mg of cell extract protein. The assay uses the same binding conditions as in BK001, i.e., no more than 75 mM total ionic strength.
5.After 20 min incubation at RT, spin at 1000 rpm for 20 sec, pipette off supernatant, and save for a gel.
6. Wash the beads 2 x 1ml in binding buffer, then resuspend in 40 ml of 2x SDS loading buffer, heat to 95°C for 2 min and load 20 ml onto into the well, and load the supernatant sample next to it.
7. Three good controls are extract alone, streptavidin beads alone, and the monomer beads versus F-actin beads, all should be run with both pellet and supernatant samples.
If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
