Actin protein (biotin): skeletal muscle

Actin protein (biotin): skeletal muscle


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. 



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.)


  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

AuthorTitleJournalYearArticle Link
Morales, E. Angelo et al.Mitotic spindle positioning protein (MISP) preferentially binds to aged F-actinJournal of Biological Chemistry2024ISSN 0021--9258
Smith, Amanda J. et al.Design and Construction of a Multi-Tiered Minimal Actin Cortex for Structural Support in Lipid Bilayer ApplicationsACS Applied Bio Materials2024ISSN 2576-6422
Henry, Conor M. et al.SYK ubiquitination by CBL E3 ligases restrains cross-presentation of dead cell-associated antigens by type 1 dendritic cellsCell reports2023ISSN 2211--1247
Whitlock, Jarred M. et al.Cell surface-bound La protein regulates the cell fusion stage of osteoclastogenesisNature Communications 2023 14:12023ISSN 2041--1723
Henty-Ridilla, Jessica L.Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) MicroscopyJoVE (Journal of Visualized Experiments)2022ISSN 1940--087X
Al Azzam, Omayma et al.Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical TweezersJoVE (Journal of Visualized Experiments)2022ISSN 1940--087X
Muresan, Camelia G. et al.F-actin architecture determines constraints on myosin thick filament motionNature Communications 2022 13:12022ISSN 2041--1723
Sasanpour, Mehrzad et al.Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and MechanicsJoVE (Journal of Visualized Experiments)2022ISSN 1940--087X
Bashirzadeh, Yashar et al.Encapsulated actomyosin patterns drive cell-like membrane shape changesiScience2022
Arbore, C. et al.α-catenin switches between a slip and an asymmetric catch bond with F-actin to cooperatively regulate cell junction fluidityNature Communications2022ISSN 2041-1723
Giampazolias, Evangelos et al.Secreted gelsolin inhibits DNGR-1-dependent cross-presentation and cancer immunityCell2021ISSN 1097-4172
Tripathi, Ananya et al.Myosin-specific adaptations of in vitro fluorescence microscopy-based motility assaysJournal of Visualized Experiments2021ISSN 1940-087X
Liu, Rong et al.A binding protein regulates myosin-7a dimerization and actin bundle assemblyNature Communications2021ISSN 2041-1723
Farhadi, Leila et al.Actin and microtubule crosslinkers tune mobility and control co-localization in a composite cytoskeletal networkSoft Matter2020ISSN 1744-6848
Mei, Lin et al.Molecular mechanism for direct actin force-sensing by α-catenineLife2020ISSN 2050-084X
Francis, Madison L. et al.Non-monotonic dependence of stiffness on actin crosslinking in cytoskeleton compositesSoft Matter2019ISSN 1744-6848
Ricketts, Shea N. et al.Varying crosslinking motifs drive the mesoscale mechanics of actin-microtubule compositesScientific Reports2019ISSN 2045-2322
Wang, Weiwei et al.Actin dynamics, regulated by RhoA-LIMK-cofilin signaling, mediates rod photoreceptor axonal retraction after retinal injuryInvestigative Ophthalmology and Visual Science2019ISSN 1552-5783
Nakos, Konstantinos et al.Septin 2/6/7 complexes tune microtubule plus-end growth and EB1 binding in a concentration- And filament-dependent mannerMolecular Biology of the Cell2019ISSN 1939-4586
Burden, Daniel L. et al.Mechanically Enhancing Planar Lipid Bilayers with a Minimal Actin CortexLangmuir2018ISSN 1520-5827
Meirson, Tomer et al.Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitorsOncotarget2018ISSN 1949-2553
Charles-Orszag, Arthur et al.Adhesion to nanofibers drives cell membrane remodeling through one-dimensional wettingNature Communications2018ISSN 2041-1723
Gardini, L. et al.High-speed optical tweezers for the study of single molecular motorsMethods in Molecular Biology2018ISSN 1064-3745
Genna, Alessandro et al.Pyk2 and FAK differentially regulate invadopodia formation and function in breast cancer cellsJournal of Cell Biology2018ISSN 1540-8140
Gurmessa, Bekele et al.Nonlinear Actin Deformations Lead to Network Stiffening, Yielding, and Nonuniform Stress PropagationBiophysical Journal2017ISSN 1542-0086
Greenberg, Michael J. et al.Measuring the kinetic and mechanical properties of non-processive myosins using optical tweezersMethods in Molecular Biology2017ISSN 1064-3745
Balikov, Daniel A. et al.The nesprin-cytoskeleton interface probed directly on single nuclei is a mechanically rich systemNucleus2017ISSN 1949-1042
Valenzuela-Iglesias, A. et al.Profilin1 regulates invadopodium maturation in human breast cancer cellsEuropean Journal of Cell Biology2015ISSN 1618-1298
Mi, Na et al.CapZ regulates autophagosomal membrane shaping by promoting actin assembly inside the isolation membraneNature Cell Biology2015ISSN 1476-4679
Wang, Y et al.Fluorescence imaging with one-nanometer accuracy (FIONA)JoVE (Journal of …2014Article Link
Beausang, John F. et al.Tilting and Wobble of Myosin V by High-Speed Single-Molecule Polarized Fluorescence MicroscopyBiophysical Journal2013ISSN 0006--3495
Shimamura, Shintaro et al.The Src substrate SKAP2 regulates actin assembly by interacting with WAVE2 and cortactin proteinsJournal of Biological Chemistry2013ISSN 0021-9258
Patsialou, Antonia et al.Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumorsIntraVital2013Article Link
Choi, Dong Shin et al.Dual transport systems based on hybrid nanostructures of microtubules and actin filamentsSmall2011ISSN 1613-6829
Van Der Gucht, Jasper et al.Stress release drives symmetry breaking for actin-based movementProceedings of the National Academy of Sciences of the United States of America2005ISSN 0027-8424
Posern, Guido et al.Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MALThe EMBO Journal2004PMID 15385960
Ono, Shoichiro et al.Microscopic evidence that actin-interacting protein 1 actively disassembles actin-depolymerizing factor/Cofilin-bound actin filamentsThe Journal of biological chemistry2004ISSN 0021--9258


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.




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