Actin protein (pyrene labeled): rabbit skeletal muscle

Actin protein (pyrene labeled): rabbit skeletal muscle
$0.00

Material

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 between 0.4-0.6 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.  

 

Purity

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).

Applications

  Application  Reference

Ubiquitin PTM Events Regulate Actin Dynamics

  1, 2

ABP Structure and Function Studies

  3, 4, 5

Actin-Focused Cancer Targeting Mechanisms

  6, 7, 8, 9

Investigation of Novel Actin Binding Molecules

  10, 11

Small GTPase signaling pathways effect on actin dynamics

  12, 13, 14, 15

Cytoskeletal regulation by microbes – bioengineering new research tools

  16, 17, 18

 

Figure 1: Pyrene Muscle Actin Protein Purity Determination.          

ap05gel

Legend-Fig. 1: 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).  Mark12 molecular weight markers are from Invitrogen.

 

 

Figure 2: Fluorescence enhancement during pyrene actin polymerization.

ap05_-_fig2

Legend-Fig. 2: Pyrene muscle actin was polymerized in duplicate wells of a 96 well plate according to the method. 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.

Biological Activity Assay

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 pyrene F-actin has a 7-12 fold fluorescent enhancement over non-polymerized pyrene G-actin.  A typical polymerization fluorescent enhancement curve is shown in Figure 2.

 

Application References

1-  Ubiquitin-dependent remodeling of the actin cytoskeleton drives cell fusion. 2021. Rodriguez-Perez F. et al. Dev Cell. 56 (5), P588-601

2-  Multi-monoubiquitylation controls VASP-mediated actin dynamics. 2024. McCormick L. et al. JCS. 137 (2): jcs261527

3-  Structural basis underlying specific biochemical activities of non-muscle tropomyosin isoforms. 2023. Selvaraj M. et al. Cell Reports. 42 (1), 111900

4-  Phase separation of an actin nucleator by junctional microtubules regulates epithelial function. 2023. Tsukita K. et al. Sci Adv. 9 (7). 

5-  Mechanisms underlying distinct subcellular localization and regulation of epithelial long myosin light-chain kinase splice variants. 2024. Chanez-Paredes S. et al. JBC. 300 (2), 105643

6-  Intermittent F-actin Perturbations by Magnetic Fields Inhibit Breast Cancer Metastasis. 2023. Ji X. et al. Research. 6, 0080

7-  CdSe/ZnS Quantum Dots’ Impact on In Vitro Actin Dynamics. 2024. Chand A. et al. Int. J. Mol. Sci. 25 (8), 4179

8-  An actin filament branching surveillance system regulates cell cycle progression, cytokinesis and primary ciliogenesis. 2023. Cao M. et al. Nature Commun. 14, 1687

9-  Cleavage of tropomodulin-3 by asparagine endopeptidase promotes cancer malignancy by actin remodeling and SND1/RhoA signaling. 2022. Chen B. et al. J Exp Clin Cancer Res. 41, 209

10-  Discovery of an F-actin–binding small molecule serving as a fluorescent probe and a scaffold for functional probes. 2021. Takagi T. et al. Sci Adv. 7 (47)

11-  Modulating dynamics and function of nuclear actin with synthetic bicyclic peptide. 2020. Machida N. et al. J of Biochem. 169 (3), 295-302

12-  ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization. 2023. Bogucka-Janczi K. et al. eLife. doi.org/10.7554/eLife.85167

13-  MICAL1 activation by PAK1 mediates actin filament disassembly. 2022. McGarry D. et al. Cell Reports. 41 (1), 111442

14-  Dual control of formin-nucleated actin assembly by the chromatin and ER in mouse oocytes. 2022. Wang H. et al. Curr Biol. 32 (18), p4013-4024

15-  Rac1 promotes kidney collecting duct integrity by limiting actomyosin activity. 2021. Bock F. et al. JCB. 220 (11), e202103080

16-  ActuAtor, a Listeria-inspired molecular tool for physical manipulation of intracellular organizations through de novo actin polymerization. 2023. Nakamura H. et al. Cell Reports. 42 (10), 113089

17-  Alloxan Disintegrates the Plant Cytoskeleton and Suppresses mlo-Mediated Powdery Mildew Resistance. 2019. Wu H. et al. Plant & Cell Phys. 61 (3), p505-518

18-  Synthesis and characterization of semisynthetic analogs of the antifungal occidiofungin. 2022. Geng M. et al. Front in Microb.  13, doi.org/10.3389/fmicb.2022.1056453

 

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
McCormick, Laura E. et al.Multi-monoubiquitylation controls VASP-mediated actin dynamicsJournal of Cell Science2024ISSN 1477-9137
Selvaraj, Muniyandi et al.Structural basis underlying specific biochemical activities of non-muscle tropomyosin isoformsCell reports2023ISSN 2211--1247
Nakamura, Hideki et al.ActuAtor, a Listeria-inspired molecular tool for physical manipulation of intracellular organizations through de novo actin polymerizationCell reports2023ISSN 2211--1247
Tsukita, Kazuto et al.Phase separation of an actin nucleator by junctional microtubules regulates epithelial functionScience Advances2023ISSN 2375-2548
Cao, Muqing et al.An actin filament branching surveillance system regulates cell cycle progression, cytokinesis and primary ciliogenesisNature Communications 2023 14:12023ISSN 2041--1723
Ji, Xinmiao et al.Intermittent F-actin Perturbations by Magnetic Fields Inhibit Breast Cancer MetastasisResearch2023ISSN 2639-5274
Bogucka-Janczi, Katarzyna et al.ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization.eLife2023ISSN 2050--084X
Selvaraj, Muniyandi et al.Structural basis underlying specific biochemical activities of non-muscle tropomyosin isoformsCell Reports2023
McGarry, David J. et al.MICAL1 activation by PAK1 mediates actin filament disassemblyCell Reports2022
Machida, Nanako et al.Modulating dynamics and function of nuclear actin with synthetic bicyclic peptidesJournal of biochemistry2021ISSN 1756-2651
Rodríguez-Pérez, Fernando et al.Ubiquitin-dependent remodeling of the actin cytoskeleton drives cell fusionDevelopmental Cell2021ISSN 1878-1551
Wu, Hongpo et al.Alloxan disintegrates the plant cytoskeleton and suppresses Mlo-mediated powdery mildew resistancePlant and Cell Physiology2020ISSN 1471-9053
Ranieri, Marianna et al.Green olive leaf extract (OLE) provides cytoprotection in renal cells exposed to low doses of cadmiumPLoS ONE2019ISSN 1932-6203
Zhang, Min et al.Axonogenesis Is Coordinated by Neuron-Specific Alternative Splicing Programming and Splicing Regulator PTBP2Neuron2019ISSN 1097-4199
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 cellsInternational Journal of Oncology2019ISSN 1791-2423
Antoku, Susumu et al.ERK1/2 Phosphorylation of FHOD Connects Signaling and Nuclear Positioning Alternations in Cardiac LaminopathyDevelopmental Cell2019ISSN 1878-1551
Schaffer, Ashleigh E. et al.Biallelic loss of human CTNNA2, encoding αN-catenin, leads to ARP2/3 complex overactivity and disordered cortical neuronal migrationNature Genetics2018ISSN 1546-1718
Almeida-Souza, Leonardo et al.A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated PitsCell2018ISSN 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 cablesMolecular Biology of the Cell2018ISSN 1939-4586
Patel, Vaibhav B. et al.PI3Kα-regulated gelsolin activity is a critical determinant of cardiac cytoskeletal remodeling and heart diseaseNature Communications2018ISSN 2041-1723
Sun, He et al.Profilin Negatively Regulates Formin-Mediated Actin Assembly to Modulate PAMP-Triggered Plant ImmunityCurrent Biology2018ISSN 0960-9822
Sun, He et al.Purification of Globular Actin from Rabbit Muscle and Pyrene Fluorescent Assays to Investigate Actin Dynamics in vitroBio-Protocol2018ISSN 2331--8325
Khatra, Harleen et al.Hedgehog Antagonist Pyrimidine–Indole Hybrid Molecule Inhibits Ciliogenesis through Microtubule DestabilisationChemBioChem2018ISSN 1439-7633
Zhao, Miao et al.Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerizationOncotarget2017ISSN 1949-2553
Cui, Jin et al.Leptolyngbyolides, Cytotoxic Macrolides from the Marine Cyanobacterium Leptolyngbya sp.: Isolation, Biological Activity, and Catalytic Asymmetric Total SynthesisChemistry - A European Journal2017ISSN 1521-3765
Currier, Mark A. et al.Identification of cancer-targeted tropomyosin inhibitors and their synergy with microtubule drugsMolecular Cancer Therapeutics2017ISSN 1538-8514
Dräger, Nina M et al. Bin1 directly remodels actin dynamics through its BAR domain EMBO reports2017ISSN 1469--221X
Yoon, Jimok et al.Amplification of F-Actin Disassembly and Cellular Repulsion by Growth Factor SignalingDevelopmental Cell2017ISSN 1878-1551
Jung, Goeh et al.V-1 regulates Capping Protein activity in vivoProceedings of the National Academy of Sciences of the United States of America2016ISSN 1091-6490
Adams, Gregory et al.The microtubule plus end tracking protein TIP150 interacts with cortactin to steer directional cell migrationJournal of Biological Chemistry2016ISSN 1083-351X
Walrant, Astrid et al.Triggering actin polymerization in xenopus egg extracts from phosphoinositide-containing lipid bilayersMethods in Cell Biology2015ISSN 0091-679X
Kremneva, Elena et al.Cofilin-2 controls actin filament length in muscle sarcomeresDevelopmental Cell2014ISSN 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 adhesionsMolecular Biology of the Cell2013ISSN 1059-1524
Zucchini, Daniela et al.Kinetic and spectroscopic characterization of the putative monooxygenase domain of human MICAL-1Archives of Biochemistry and Biophysics2011ISSN 0003-9861
Rapier, Rebecca et al.The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cellsCancer Cell International2010ISSN 1475-2867
Hao, Yuan Kueha et al.Dynamic stabilization of actin filamentsProceedings of the National Academy of Sciences of the United States of America2008ISSN 1091-6490
Allingham, John S. et al.A Structural Basis for Regulation of Actin Polymerization by PectenotoxinsJournal of Molecular Biology2007ISSN 0022-2836
Leng, Yan et al.Abelson-interactor-1 promotes WAVE2 membrane translocation and Abelson-mediated tyrosine phosphory lation required for WAVE2 activationProceedings of the National Academy of Sciences of the United States of America2005ISSN 0027-8424
Allingham, John S. et al.Structures of microfilament destabilizing toxins bound to actin provide insight into toxin design and activityProceedings of the National Academy of Sciences of the United States of America2005ISSN 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 membranePLoS Biology2004ISSN 1544-9173
Balcer, Heath I. et al.Coordinated Regulation of Actin Filament Turnover by a High-Molecular-Weight Srv2/CAP Complex, Cofilin, Profilin, and Aip12003PMID 14680631
Humphries, Christine L. et al.Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin2002ISSN 0021--9525
Wahlström, Gudrun et al.Twinfilin is required for actin-dependent developmental processes in DrosophilaJournal of Cell Biology2001ISSN 0021--9525
Uruno, Takehito et al.Activation of Arp2/3 complex-mediated actin polymerization by cortactinNature Cell Biology 2001 3:32001ISSN 1476--4679
Duncan, Mara C. et al.Yeast Eps15-like endocytic protein, Pan1p, activates the Arp2/3 complexNature Cell Biology 2001 3:72001ISSN 1476--4679

 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 tservice@cytoskeleton.com.