Product Uses Include
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.
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).
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.
|Arbore, C. et al.||α-catenin switches between a slip and an asymmetric catch bond with F-actin to cooperatively regulate cell junction fluidity||Nature Communications||2022||ISSN 2041-1723|
|Liu, Rong et al.||A binding protein regulates myosin-7a dimerization and actin bundle assembly||Nature Communications||2021||ISSN 2041-1723|
|Tripathi, Ananya et al.||Myosin-specific adaptations of in vitro fluorescence microscopy-based motility assays||Journal of Visualized Experiments||2021||ISSN 1940-087X|
|Giampazolias, Evangelos et al.||Secreted gelsolin inhibits DNGR-1-dependent cross-presentation and cancer immunity||Cell||2021||ISSN 1097-4172|
|Bashirzadeh, Yashar et al.||Encapsulated Actomyosin Patterns Drive Cell-Like Membrane Shape Changes||SSRN Electronic Journal||2021||Article Link|
|Mei, Lin et al.||Molecular mechanism for direct actin force-sensing by α-catenin||eLife||2020||ISSN 2050-084X|
|Farhadi, Leila et al.||Actin and microtubule crosslinkers tune mobility and control co-localization in a composite cytoskeletal network||Soft Matter||2020||ISSN 1744-6848|
|Wang, Weiwei et al.||Actin dynamics, regulated by RhoA-LIMK-cofilin signaling, mediates rod photoreceptor axonal retraction after retinal injury||Investigative Ophthalmology and Visual Science||2019||ISSN 1552-5783|
|Francis, Madison L. et al.||Non-monotonic dependence of stiffness on actin crosslinking in cytoskeleton composites||Soft Matter||2019||ISSN 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 manner||Molecular Biology of the Cell||2019||ISSN 1939-4586|
|Ricketts, Shea N. et al.||Varying crosslinking motifs drive the mesoscale mechanics of actin-microtubule composites||Scientific Reports||2019||ISSN 2045-2322|
|Meirson, Tomer et al.||Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors||Oncotarget||2018||ISSN 1949-2553|
|Burden, Daniel L. et al.||Mechanically Enhancing Planar Lipid Bilayers with a Minimal Actin Cortex||Langmuir||2018||ISSN 1520-5827|
|Charles-Orszag, Arthur et al.||Adhesion to nanofibers drives cell membrane remodeling through one-dimensional wetting||Nature Communications||2018||ISSN 2041-1723|
|Gardini, L. et al.||High-speed optical tweezers for the study of single molecular motors||Methods in Molecular Biology||2018||ISSN 1064-3745|
|Genna, Alessandro et al.||Pyk2 and FAK differentially regulate invadopodia formation and function in breast cancer cells||Journal of Cell Biology||2018||ISSN 1540-8140|
|Gurmessa, Bekele et al.||Nonlinear Actin Deformations Lead to Network Stiffening, Yielding, and Nonuniform Stress Propagation||Biophysical Journal||2017||ISSN 1542-0086|
|Greenberg, Michael J. et al.||Measuring the kinetic and mechanical properties of non-processive myosins using optical tweezers||Methods in Molecular Biology||2017||ISSN 1064-3745|
|Balikov, Daniel A. et al.||The nesprin-cytoskeleton interface probed directly on single nuclei is a mechanically rich system||Nucleus||2017||ISSN 1949-1042|
|Valenzuela-Iglesias, A. et al.||Profilin1 regulates invadopodium maturation in human breast cancer cells||European Journal of Cell Biology||2015||ISSN 1618-1298|
|Mi, Na et al.||CapZ regulates autophagosomal membrane shaping by promoting actin assembly inside the isolation membrane||Nature Cell Biology||2015||ISSN 1476-4679|
|Wang, Y et al.||Fluorescence imaging with one-nanometer accuracy (FIONA)||JoVE (Journal of …||2014||Article Link|
|Patsialou, Antonia et al.||Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors||IntraVital||2013||Article Link|
|Shimamura, Shintaro et al.||The Src substrate SKAP2 regulates actin assembly by interacting with WAVE2 and cortactin proteins||Journal of Biological Chemistry||2013||ISSN 0021-9258|
|Beausang, John F. et al.||Tilting and Wobble of Myosin V by High-Speed Single-Molecule Polarized Fluorescence Microscopy||Biophysical Journal||2013|
|Choi, Dong Shin et al.||Dual transport systems based on hybrid nanostructures of microtubules and actin filaments||Small||2011||ISSN 1613-6829|
|Van Der Gucht, Jasper et al.||Stress release drives symmetry breaking for actin-based movement||Proceedings of the National Academy of Sciences of the United States of America||2005||ISSN 0027-8424|
|Ono, Shoichiro et al.||Microscopic evidence that actin-interacting protein 1 actively disassembles actin-depolymerizing factor/Cofilin-bound actin filaments||The Journal of biological chemistry||2004||ISSN 0021--9258|
|Posern, Guido et al.||Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MAL||The EMBO Journal||2004||PMID 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 firstname.lastname@example.org