Product Uses Include
Purified rabbit muscle actin (Cat. # AKL99) has been modified to contain covalently linked rhodamine at random surface lysine residues. An activated ester of rhodamine is used to label the protein. The labeling stoichiometry has been determined to be 1-2 dyes per actin monomer. Rhodamine labeled rabbit muscle actin has an approximate molecular weight of 43 kDa, and is supplied as a pink lyophilized powder. The lyophilized protein is stable for 6 months when stored desiccated to <10% humidity at 4°C. The protein should be reconstituted to 10 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.
Rhodamine actin from a non-muscle source is also available (Cat. # APHR).
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% polyacrylamide gel. AR05 rhodamine muscle actin was found to be >99% pure (see Figure 1).
Figure 1. A 100 µg sample of rhodamine muscle actin (molecular weight approx. 43 kDa) was separated by electrophoresis in a 12% SDS-PAGE system, and stained with Coomassie Blue. Protein quantitation was performed with the Precision Red Protein Assay Reagent (Cat. # ADV02).
The biological activity of rhodamine muscle actin can be determined by its ability to efficiently polymerize into filaments in vitro and separate from unpolymerized components in a spin down assay. Stringent quality control ensures that 90% of the labeled muscle actin can polymerized in this assay, which is similar to the unlabeled product (Cat. # AKL99)
|McGrath, Jamis et al.||Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilin||Current Biology||2021||ISSN 1879-0445|
|Kučera, Ondřej et al.||Anillin propels myosin-independent constriction of actin rings||Nature Communications||2021||ISSN 2041-1723|
|Giampazolias, Evangelos et al.||Secreted gelsolin inhibits DNGR-1-dependent cross-presentation and cancer immunity||Cell||2021||ISSN 1097-4172|
|Sun, Xiaoyu et al.||Mechanosensing through Direct Binding of Tensed F-Actin by LIM Domains||Developmental Cell||2020||ISSN 1878-1551|
|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|
|Padmanabhan, Krishnanand et al.||Thymosin β4 is essential for adherens junction stability and epidermal planar cell polarity||Development (Cambridge)||2020||ISSN 1477-9129|
|Mei, Lin et al.||Molecular mechanism for direct actin force-sensing by α-catenin||eLife||2020||ISSN 2050-084X|
|Skruber, Kristen et al.||Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge||Current Biology||2020||ISSN 1879-0445|
|Lin, Shan Shan et al.||Dynamin-2 Regulates Postsynaptic Cytoskeleton Organization and Neuromuscular Junction Development||Cell Reports||2020||ISSN 2211-1247|
|Beutel, Oliver et al.||Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions||Cell||2019||ISSN 1097-4172|
|Chan, Byron et al.||Adseverin, an actin binding protein, regulates articular chondrocyte phenotype||Journal of Tissue Engineering and Regenerative Medicine||2019||ISSN 1932-7005|
|Burden, Daniel L. et al.||Mechanically Enhancing Planar Lipid Bilayers with a Minimal Actin Cortex||Langmuir||2018||ISSN 1520-5827|
|Zeng, Menglong et al.||Reconstituted Postsynaptic Density as a Molecular Platform for Understanding Synapse Formation and Plasticity||Cell||2018||ISSN 1097-4172|
|Silván, Unai et al.||Contributions of the lower dimer to supramolecular actin patterning revealed by TIRF microscopy||Journal of Structural Biology||2016||ISSN 1095-8657|
|Jiang, Hongwei et al.||Adseverin plays a role in osteoclast differentiation and periodontal disease-mediated bone loss||FASEB Journal||2015||ISSN 1530-6860|
|Ramamurthy, Bhagavathi et al.||Plus-end directed myosins accelerate actin filament sliding by single-headed myosin VI||Cytoskeleton||2012||ISSN 1949-3584|
|Del Duca, Stefano et al.||Effects of post-translational modifications catalysed by pollen transglutaminase on the functional properties of microtubulesand actin filaments||Biochemical Journal||2009||ISSN 1470-8728|
|Klaavuniemi, Tuula et al.||Caenorhabditis elegans gelsolin-like protein 1 is a novel actin filament-severing protein with four gelsolin-like repeats||Journal of Biological Chemistry||2008||ISSN 0021-9258|
Question 1: What is the best way to store actin proteins to insure maximum stability and shelf-life?
Answer 1: Cytoskeleton provides all actin proteins as lyophilized powders so that they can be shipped at room temperature. Upon receipt, the lyophilized powders should be stored at 4°C in a sealed container with desiccant. It is important to monitor the freshness of the desiccant and insure that it continues to absorb moisture to protect the lyophilized actins. With proper storage, the lyophilized actins are guaranteed to be stable for 6 months. Alternatively, actins can be immediately resuspended at the concentration recommended, aliquoted, snap-frozen in liquid nitrogen and stored at -70°C. When thawing frozen aliquots, it is important to thaw rapidly in a room temperature water bath.
Question 2: What is the best way to store F-actin after polymerizing?
Answer 2: G-actin is stable for two days at 4°C and requires a divalent cation, pH 6.5 - 8.0 and ATP for stability. F-actin is stable and can be stored at 4°C for 1-2 weeks. F-actin requires ATP (0.2 mM) and Mg2+ (2 mM) for stability and is unstable below pH 6.5 and above pH 8.5. F-actin is not stable to freezing. F-actin can be transferred to a variety of buffers (e.g. HEPES, phosphate, etc) without detrimental effects. We recommend the addition of antibacterial agents such as 100 μg/ml ampicillin and 10 μg/ml chloramphenicol when storing F-actin at 4°C.
Question 3: Filters for visualizing rhodamine signal?
Answer 3: The excitation filter should be set at 535 nm and the emission filter at 585 nm.
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