Actin protein (pyrene labeled): rabbit skeletal muscle
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.
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.
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
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.
