Actin protein (biotin): skeletal muscle

Actin protein (biotin): skeletal muscle
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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. (see Figure 1.). No free biotin is apparent in the final product.

Purity determination of AB07

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.

ab07lblot

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.

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

AuthorTitleJournalYearArticle Link
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
Liu, Rong et al.A binding protein regulates myosin-7a dimerization and actin bundle assemblyNature Communications2021ISSN 2041-1723
Tripathi, Ananya et al.Myosin-specific adaptations of in vitro fluorescence microscopy-based motility assaysJournal of Visualized Experiments2021ISSN 1940-087X
Giampazolias, Evangelos et al.Secreted gelsolin inhibits DNGR-1-dependent cross-presentation and cancer immunityCell2021ISSN 1097-4172
Bashirzadeh, Yashar et al.Encapsulated Actomyosin Patterns Drive Cell-Like Membrane Shape ChangesSSRN Electronic Journal2021Article Link
Mei, Lin et al.Molecular mechanism for direct actin force-sensing by α-catenineLife2020ISSN 2050-084X
Farhadi, Leila et al.Actin and microtubule crosslinkers tune mobility and control co-localization in a composite cytoskeletal networkSoft Matter2020ISSN 1744-6848
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
Francis, Madison L. et al.Non-monotonic dependence of stiffness on actin crosslinking in cytoskeleton compositesSoft Matter2019ISSN 1744-6848
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
Ricketts, Shea N. et al.Varying crosslinking motifs drive the mesoscale mechanics of actin-microtubule compositesScientific Reports2019ISSN 2045-2322
Meirson, Tomer et al.Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitorsOncotarget2018ISSN 1949-2553
Burden, Daniel L. et al.Mechanically Enhancing Planar Lipid Bilayers with a Minimal Actin CortexLangmuir2018ISSN 1520-5827
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
Patsialou, Antonia et al.Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumorsIntraVital2013Article Link
Shimamura, Shintaro et al.The Src substrate SKAP2 regulates actin assembly by interacting with WAVE2 and cortactin proteinsJournal of Biological Chemistry2013ISSN 0021-9258
Beausang, John F. et al.Tilting and Wobble of Myosin V by High-Speed Single-Molecule Polarized Fluorescence MicroscopyBiophysical Journal2013
Choi, Dong Shin et al.Dual transport systems based on hybrid nanostructures of microtubules and actin filamentsSmall2011ISSN 1613-6829
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Posern, Guido et al.Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MALThe EMBO Journal2004PMID 15385960

 

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