Product Uses Include
Purified rabbit muscle actin (Cat. # AKL99) has been modified to contain covalently linked pyrene at the cysteine 374 residue. An N-(1-pyrene) iodoacetamide is used to label the actin protein. Pyrene labeling stoichiometry has been determined to be 0.6 dyes per actin monomer. Pyrene labeled rabbit muscle actin has an approximate molecular weight of 43 kDa, and is supplied as a white lyophilized powder. The lyophilized protein is st able for 6 months when stored desiccated to <10% humidity at 4°C. The protein should be reconstituted to 20 mg/ml with distilled water; it 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 4-20% gradient polyacrylamide gel. Pyrene muscle actin is >99% pure (see Figure 1).
Figure 1. Pyrene Muscle Actin Protein Purity Determination. A 100 µg sample of pyrene muscle actin (molecular weight approx. 43 kDa) was separated by electrophoresis in a 4-20% SDS-PAGE system, and stained with Coomassie Blue. Protein quantitation was determined with the Precision Red Protein Assay Reagent (Cat. # ADV02).
The fluorescent signal of monomer pyrene actin is enhanced during its polymerization into filaments, making it an ideal tool for monitoring actin filament formation. Stringent quality control ensures that AP05 pyrene F-actin has a 7-12 fold fluorescent enhancement over non-polymerized pyrene G-actin (See Fig 2).
Figure 2. Fluorescence enhancement during pyrene actin polymerization. Pyrene muscle actin was polymerized in duplicate wells of a 96-well plate by the addition of Actin Polymerization Buffer (Cat. # BSA02). The fluorescent signal was scanned every 30 s for 1 h. Polymerized pyrene F-actin shows a 10 fold fluorescent enhancement over non-polymerized pyrene G-actin and buffer control.
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 email@example.com
|Selvaraj, Muniyandi et al.||Structural basis underlying specific biochemical activities of non-muscle tropomyosin isoforms||Cell Reports||2023|
|McGarry, David J. et al.||MICAL1 activation by PAK1 mediates actin filament disassembly||Cell Reports||2022|
|Machida, Nanako et al.||Modulating dynamics and function of nuclear actin with synthetic bicyclic peptides||Journal of biochemistry||2021||ISSN 1756-2651|
|Rodríguez-Pérez, Fernando et al.||Ubiquitin-dependent remodeling of the actin cytoskeleton drives cell fusion||Developmental Cell||2021||ISSN 1878-1551|
|Wu, Hongpo et al.||Alloxan disintegrates the plant cytoskeleton and suppresses Mlo-mediated powdery mildew resistance||Plant and Cell Physiology||2020||ISSN 1471-9053|
|Zhang, Min et al.||Axonogenesis Is Coordinated by Neuron-Specific Alternative Splicing Programming and Splicing Regulator PTBP2||Neuron||2019||ISSN 1097-4199|
|Ranieri, Marianna et al.||Green olive leaf extract (OLE) provides cytoprotection in renal cells exposed to low doses of cadmium||PLoS ONE||2019||ISSN 1932-6203|
|Abe, Tadashi et al.||Phosphorylation of cortactin by cyclin-dependent kinase 5 modulates actin bundling by the dynamin 1-cortactin ring-like complex and formation of filopodia and lamellipodia in NG108-15 glioma-derived cells||International Journal of Oncology||2019||ISSN 1791-2423|
|Antoku, Susumu et al.||ERK1/2 Phosphorylation of FHOD Connects Signaling and Nuclear Positioning Alternations in Cardiac Laminopathy||Developmental Cell||2019||ISSN 1878-1551|
|Khatra, Harleen et al.||Hedgehog Antagonist Pyrimidine–Indole Hybrid Molecule Inhibits Ciliogenesis through Microtubule Destabilisation||ChemBioChem||2018||ISSN 1439-7633|
|Schaffer, Ashleigh E. et al.||Biallelic loss of human CTNNA2, encoding αN-catenin, leads to ARP2/3 complex overactivity and disordered cortical neuronal migration||Nature Genetics||2018||ISSN 1546-1718|
|Almeida-Souza, Leonardo et al.||A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits||Cell||2018||ISSN 1097-4172|
|Shin, Myungjoo et al.||Yeast Aim21/Tda2 both regulates free actin by reducing barbed end assembly and forms a complex with Cap1/Cap2 to balance actin assembly between patches and cables||Molecular Biology of the Cell||2018||ISSN 1939-4586|
|Sun, He et al.||Purification of Globular Actin from Rabbit Muscle and Pyrene Fluorescent Assays to Investigate Actin Dynamics in vitro||Bio-Protocol||2018||ISSN 2331--8325|
|Sun, He et al.||Profilin Negatively Regulates Formin-Mediated Actin Assembly to Modulate PAMP-Triggered Plant Immunity||Current Biology||2018||ISSN 0960-9822|
|Patel, Vaibhav B. et al.||PI3Kα-regulated gelsolin activity is a critical determinant of cardiac cytoskeletal remodeling and heart disease||Nature Communications||2018||ISSN 2041-1723|
|Zhao, Miao et al.||Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization||Oncotarget||2017||ISSN 1949-2553|
|Dräger, Nina M et al.||Bin1 directly remodels actin dynamics through its BAR domain||EMBO reports||2017||ISSN 1469--221X|
|Yoon, Jimok et al.||Amplification of F-Actin Disassembly and Cellular Repulsion by Growth Factor Signaling||Developmental Cell||2017||ISSN 1878-1551|
|Cui, Jin et al.||Leptolyngbyolides, Cytotoxic Macrolides from the Marine Cyanobacterium Leptolyngbya sp.: Isolation, Biological Activity, and Catalytic Asymmetric Total Synthesis||Chemistry - A European Journal||2017||ISSN 1521-3765|
|Currier, Mark A. et al.||Identification of cancer-targeted tropomyosin inhibitors and their synergy with microtubule drugs||Molecular Cancer Therapeutics||2017||ISSN 1538-8514|
|Adams, Gregory et al.||The microtubule plus end tracking protein TIP150 interacts with cortactin to steer directional cell migration||Journal of Biological Chemistry||2016||ISSN 1083-351X|
|Jung, Goeh et al.||V-1 regulates Capping Protein activity in vivo||Proceedings of the National Academy of Sciences of the United States of America||2016||ISSN 1091-6490|
|Walrant, Astrid et al.||Triggering actin polymerization in xenopus egg extracts from phosphoinositide-containing lipid bilayers||Methods in Cell Biology||2015||ISSN 0091-679X|
|Kremneva, Elena et al.||Cofilin-2 controls actin filament length in muscle sarcomeres||Developmental Cell||2014||ISSN 1878-1551|
|Arora, P. D. et al.||Collagen remodeling by phagocytosis is determined by collagen substrate topology and calcium-dependent interactions of gelsolin with nonmuscle myosin IIA in cell adhesions||Molecular Biology of the Cell||2013||ISSN 1059-1524|
|Zucchini, Daniela et al.||Kinetic and spectroscopic characterization of the putative monooxygenase domain of human MICAL-1||Archives of Biochemistry and Biophysics||2011||ISSN 0003-9861|
|Rapier, Rebecca et al.||The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cells||Cancer Cell International||2010||ISSN 1475-2867|
|Hao, Yuan Kueha et al.||Dynamic stabilization of actin filaments||Proceedings of the National Academy of Sciences of the United States of America||2008||ISSN 1091-6490|
|Allingham, John S. et al.||A Structural Basis for Regulation of Actin Polymerization by Pectenotoxins||Journal of Molecular Biology||2007||ISSN 0022-2836|
|Allingham, John S. et al.||Structures of microfilament destabilizing toxins bound to actin provide insight into toxin design and activity||Proceedings of the National Academy of Sciences of the United States of America||2005||ISSN 0027-8424|
|Leng, Yan et al.||Abelson-interactor-1 promotes WAVE2 membrane translocation and Abelson-mediated tyrosine phosphory lation required for WAVE2 activation||Proceedings of the National Academy of Sciences of the United States of America||2005||ISSN 0027-8424|
|Holaska, James M. et al.||Emerin caps the pointed end of actin filaments: Evidence for an actin cortical network at the nuclear inner membrane||PLoS Biology||2004||ISSN 1544-9173|
|Balcer, Heath I. et al.||Coordinated Regulation of Actin Filament Turnover by a High-Molecular-Weight Srv2/CAP Complex, Cofilin, Profilin, and Aip1||2003||PMID 14680631|
|Humphries, Christine L. et al.||Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin||2002||ISSN 0021--9525|
|Duncan, Mara C. et al.||Yeast Eps15-like endocytic protein, Pan1p, activates the Arp2/3 complex||Nature Cell Biology 2001 3:7||2001||ISSN 1476--4679|
|Uruno, Takehito et al.||Activation of Arp2/3 complex-mediated actin polymerization by cortactin||Nature Cell Biology 2001 3:3||2001||ISSN 1476--4679|
|Wahlström, Gudrun et al.||Twinfilin is required for actin-dependent developmental processes in Drosophila||Journal of Cell Biology||2001||ISSN 0021--9525|
Question 1: When performing the polymerization assay using pyrene-labeled actin (Cat. # AP05), the fluorescence signal of the globular actin in G-buffer increased over time, so that the 0 value was already rather high.
Answer 1: After preparing actin at a concentration of 0.4 mg/ml for polymerization (from either fresh stocks or frozen aliquots), it is important to incubate the actin on ice for 1 hour to depolymerize any actin polymers that might have formed during storage. For an even cleaner sample of G-actin, the samples can be centrifuged (100,000 x g) for 60 min to absolutely insure the removal of residual actin oligomers and nucleating centers. After centrifugation, use the top 80% of the supernatant as your G-actin stock. This will eliminate any changes in fluorescence signal associated with G-actin.
Question 2: During my polymerization reaction, the growth phase and steady-state levels of F-actin produced are lower than what is shown in the manual. Why?
Answer 2: One of the most common causes of reduced fluorescence intensity signal is non-optimal machine settings. Please check that the machine’s settings are similar to these:
Measurement type: Kinetic 120 cycles, 60 sec interval time
Fluorescence wavelengths: Ex. 350 or 360 +/- 20 nm
Em. 407 or 410 +/- 10 nm or 420 +/- 20 nm
Gain: 100 (on a scale of 0-120, where 120 is the highest)
Reads per well: 1
Fluorescence reading from: Top
Also, when samples are not being measured in the fluorimeter, the excitation beam should be shuttered to prevent the beam from bleaching the pyrene fluorescence. When measuring the fluorescence, the shutter should be opened for no longer than 7 sec every 30 sec for 1 h.
If you have any questions concerning this product, please contact our Technical Service department at firstname.lastname@example.org.