Product Uses Include
Non-muscle actin has been purified from human platelets. Each unit of platelets used in the preparation of non-muscle actin has been found to be non-reactive by an FDA approved test for HBsAg, HBcAb, HIV-1/2 ab, HIV-1 RNA, HTLV I/II ab, HCV ab, HCV RNA, and syphilis. Each unit of platelets has been ALT tested with results less than an established cutoff. The isotype composition of non-muscle actin is 85% β-actin and 15% γ-actin. Non-muscle actin has an approximate molecular weight of 43 kDa. The product is provided as a lyophilized white 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.
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% polyacrylamide gel. APHL99 consists of >99% pure non-muscle actin while APHL95 is >95% pure (see Figure 1).
Figure 1: Figure 1. Purities of human platelet non-muscle actin protein. 100 µg of >99% pure (APHL99) and >95% pure (APHL95) non-muscle actin were run on SDS-PAGE gels and stained with coomassie blue. The arrow indicates actin protein (~43 kDa), the arrowhead a gelsolin contaminant (~90 kDa). The minor impurities in the purified actins are predominantly actin binding proteins such as gelsolin and α-actinin. Protein quantitation was determined with the Precision Red Protein Assay Reagent (Cat. # ADV02)
The biological activity of muscle actinis determined by its ability to efficiently polymerize into filaments (F-actin) in vitro and separate from unpolymerized components in a spin down assay. Stringent quality control ensures that APHL99 produces >85% F-actin and APHL95 produces >75% F-actin in this assay.
|Selvaraj, Muniyandi et al.
|Structural basis underlying specific biochemical activities of non-muscle tropomyosin isoforms
|Hamasaki, Eriko et al.
|The Lipid-Binding Defective Dynamin 2 Mutant in Charcot-Marie-Tooth Disease Impairs Proper Actin Bundling and Actin Organization in Glomerular Podocytes
|Frontiers in Cell and Developmental Biology
|Tsai, Feng Ching et al.
|Activated I-BAR IRSp53 clustering controls the formation of VASP-actin–based membrane protrusions
|Chen, Li et al.
|Differential N-terminal processing of beta and gamma actin
|La, The Mon et al.
|Dynamin 1 is important for microtubule organization and stabilization in glomerular podocytes
|Park, Jin Suk et al.
|Mechanical regulation of glycolysis via cytoskeleton architecture
|Ergin, Volkan et al.
|Putative Coiled-Coil Domain-Dependent Autoinhibition and Alternative Splicing Determine SHTN1’s Actin-Binding Activity
|Journal of Molecular Biology
|Slater, Paula G. et al.
|XMAP215 promotes microtubule-F-actin interactions to regulate growth cone microtubules during axon guidance in Xenopus laevis
|Journal of cell science
|Zhang, Shengnan et al.
|In-cell NMR study of Tau and MARK2 phosphorylated Tau
|International Journal of Molecular Sciences
|Figard, Lauren et al.
|Cofilin-Mediated Actin Stress Response Is Maladaptive in Heat-Stressed Embryos
|Osório, Daniel S. et al.
|Crosslinking activity of non-muscle myosin II is not sufficient for embryonic cytokinesis in C. elegans
|Development (Cambridge, England)
|Antoku, Susumu et al.
|ERK1/2 Phosphorylation of FHOD Connects Signaling and Nuclear Positioning Alternations in Cardiac Laminopathy
|Silkworth, William T. et al.
|The neuron-specific formin Delphilin nucleates nonmuscle actin but does not enhance elongation
|Molecular biology of the cell
|McIntosh, Betsy B. et al.
|Opposing Kinesin and Myosin-I Motors Drive Membrane Deformation and Tubulation along Engineered Cytoskeletal Networks
|Olthoff, John T. et al.
|Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscle
|Raoux-Barbot, Dorothée et al.
|Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin
|Balchin, David et al.
|Pathway of Actin Folding Directed by the Eukaryotic Chaperonin TRiC
|Cervero, Pasquale et al.
|Lymphocyte-specific protein 1 regulates mechanosensory oscillation of podosomes and actin isoform-based actomyosin symmetry breaking
|Tsai, Feng Ching et al.
|Ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partner
|Cabrales Fontela, Yunior et al.
|Multivalent cross-linking of actin filaments and microtubules through the microtubule-associated protein Tau
|Dräger, Nina M et al.
|Bin1 directly remodels actin dynamics through its BAR domain
|Rondina, M. T. et al.
|Non-genomic activities of retinoic acid receptor alpha control actin cytoskeletal events in human platelets
|Journal of Thrombosis and Haemostasis
|Reeg, Sandra et al.
|The molecular chaperone Hsp70 promotes the proteolytic removal of oxidatively damaged proteins by the proteasome
|Free Radical Biology and Medicine
|Alqassim, Saif S. et al.
|Modulation of MICAL Monooxygenase Activity by its Calponin Homology Domain: Structural and Mechanistic Insights
|Neasta, Jeremie et al.
|Activation of the cAMP pathway induces RACK1-dependent binding of β-actin to BDNF promoter
|Belyy, Alexander et al.
|Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins
|Sobierajska, Katarzyna et al.
|Protein disulfide isomerase directly interacts with β-actin Cys374 and regulates cytoskeleton reorganization
|The Journal of biological chemistry
|Lockett, Stephen et al.
|Quantitative analysis of F-actin redistribution in astrocytoma cells treated with candidate pharmaceuticals
|Cytometry Part A
|Kremneva, Elena et al.
|Cofilin-2 controls actin filament length in muscle sarcomeres
|Lamothe, Betty et al.
|TAK1 is essential for osteoclast differentiation and is an important modulator of cell death by apoptosis and necroptosis
|Molecular and cellular biology
|Gilmore, Jamie L. et al.
|AFM Investigation of the Organization of Actin Bundles Formed by Actin-Binding Proteins
|Journal of Surface Engineered Materials and Advanced Technology
|Marat, Andrea L. et al.
|Connecdenn 3/DENND1C binds actin linking Rab35 activation to the actin cytoskeleton
|Molecular Biology of the Cell
|Gaidos, Gabriel et al.
|Structure and function analysis of the CMS/CIN85 protein family identifies actin-bundling properties and heterotypic-complex formation
|Journal of Cell Science
|Gohla, Antje et al.
|Chronophin, a novel HAD-type serine protein phosphatase, regulates cofilin-dependent actin dynamics
|Nature cell biology
|Posern, Guido et al.
|Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MAL
|The EMBO Journal
|Searles, Charles D. et al.
|Actin cytoskeleton organization and posttranscriptional regulation of endothelial nitric oxide synthase during cell growth
|Roger, Benoit et al.
|MAP2c, but not tau, binds and bundles F-actin via its microtubule binding domain
|Chew, Catherine S. et al.
|Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo
|Journal of cell science
|Kuriyama, Ryoko et al.
|CHO1, a mammalian kinesin-like protein, interacts with F-actin and is involved in the terminal phase of cytokinesis
|Journal of Cell Biology
|Kessels, Michael M. et al.
|Association of mouse actin-binding protein 1 (mAbp1/SH3P7), an Src kinase target, with dynamic regions of the cortical actin cytoskeleton in response to Rac1 activation
|Molecular biology of the cell
|Vartiainen, Maria et al.
|Mouse A6/Twinfilin Is an Actin Monomer-Binding Protein That Localizes to the Regions of Rapid Actin Dynamics
|Molecular and Cellular Biology
|Zhou, Daoguo et al.
|Role of the S. typhimurium Actin-Binding Protein SipA in Bacterial Internalization
Question 1: Do you have pyrene-labeled non-muscle actin for use in a polymerization assay?
Answer 1: Pyrene-labeled non-muscle actin has been shown to be unstable under normal storage conditions and was discontinued. To examine the polymerization of unlabeled non-muscle actin, please click here for a polymerization protocol that uses an excess of unlabeled non-muscle actin (Cat# APHL99) + a small amount of pyrene-labeled muscle actin (Cat. # AP05). The pyrene muscle actin will not polymerize efficiently on its own at the concentration used in this assay, so the reaction is dependent on unlabeled actin polymerization for F-actin formation. In this way, the pyrene-labeled muscle actin is taken up and polymerized to serve as a reporter for polymerization of the unlabeled non-muscle actin that is present at a much greater concentration.
Question 2: Are the actin products shipped as pure G-actin or a mixture of G- and F-actin?
Answer 2: Most of our actin proteins are sold in the monomer form (G-actin) because this is stable to freezing and lyophilization. That being said, on the day of the experiment, we do recommend incubating the actin on ice for 60 min before beginning the experiment to depolymerize any actin oligomers that might have formed during storage. Typically actin is first diluted to 0.4 or 0.2 mg/ml concentration and then this can be incubated on ice for 60 min to depolymerize any actin oligomers that might have formed. If you are working with an actin concentration above 0.4 mg/ml, we recommend the ice incubation followed by a high-speed centrifugation (100,000 x g) for 60 min to pellet any actin oligomers that may not have depoymerized. Remove the top 80% of the supernatant and use this as your G-actin stock. We also provide pre-formed actin filaments (Cat. # AKF99) that are shipped lyophilized and upon resuspension, the filaments are ready for use and average 5-10 microns in length.
If you have any questions concerning this product, please contact our Technical Service department at email@example.com