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Actin protein ( >99% pure, human platelet beta gamma actin )

Actin protein (>99% pure): human platelet
$0.00
SKU
APHL99
Datasheet
MSDS
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Product Uses Include

  • Identification and characterization of non-muscle beta/gamma actin binding proteins
  • In vitro actin polymerization studies
  • Antibody standard for beta actin Western blot analysis

Material
Non-muscle beta:gamma (85:15) 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 (beta) and 15% γ-actin (gamma). 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.
 

Purity
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).

Click Here for Detailed Methods
aphlgels

   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)

Biological Activity
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.

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

 

  • For a guide to performing actin polymerizations with this actin product please click here.

AuthorTitleJournalYearArticle Link
Yamada, Hiroshi et al.Direct Binding of Synaptopodin 2-Like Protein to Alpha-Actinin Contributes to Actin Bundle Formation in CardiomyocytesCells2024
Tsuji, Chisato et al.CryoET reveals actin filaments within platelet microtubulesNature Communications2024
Kumari, Reena et al.Focal adhesions contain three specialized actin nanoscale layersNature Communications 2024
Shah, Riya et al.The DIAPH3 linker specifies a β-actin network that maintains RhoA and Myosin-II at the cytokinetic furrowNature Communications2024
Reggi, Erica et al.AKAP2-anchored protein phosphatase 1 controls prostatic neuroendocrine carcinoma cell migration and invasionBiochimica et Biophysica Acta (BBA) - Molecular Basis of Disease2024
Chanez-Paredes, Sandra D. et al.Mechanisms underlying distinct subcellular localization and regulation of epithelial long myosin light-chain kinase splice variantsJournal of Biological Chemistry2024
Delaunay, Marion et al.AKAP2-anchored extracellular signal-regulated kinase 1 (ERK1) regulates cardiac myofibroblast migrationBiochimica et Biophysica Acta (BBA) - Molecular Cell Research2024
Puccini, Joseph et al.Cytoskeletal association of ATP citrate lyase controls the mechanodynamics of macropinocytosisProceedings of the National Academy of Sciences2023
Liebe, Nils L. et al.Bioinspired Membrane Interfaces: Controlling Actomyosin Architecture and ContractilityACS Applied Materials and Interfaces2023
Nietmann, Peter et al.Cytosolic actin isoforms form networks with different rheological properties that indicate specific biological functionNature Communications2023
Yao, Min et al.Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies against self-antigensJCI Insight2023
Selvaraj, Muniyandi et al.Structural basis underlying specific biochemical activities of non-muscle tropomyosin isoformsCell reports2023
Puccini, Joseph et al.Cytoskeletal association of ATP citrate lyase controls the mechanodynamics of macropinocytosisProceedings of the National Academy of Sciences of the United States of America2023
Du, William W. et al.Nuclear Actin Polymerization Regulates Cell Epithelial-Mesenchymal TransitionAdvanced Science2023
Martin, Jose L. et al.Actin Isoform Composition and Binding Factors Fine-Tune Regulatory Impact of Mical EnzymesInternational Journal of Molecular Sciences2023
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 PodocytesFrontiers in Cell and Developmental Biology2022
Tsai, Feng Ching et al.Activated I-BAR IRSp53 clustering controls the formation of VASP-actin–based membrane protrusionsScience Advances2022
Chen, Li et al.Differential N-terminal processing of beta and gamma actiniScience2022
La, The Mon et al.Dynamin 1 is important for microtubule organization and stabilization in glomerular podocytesFASEB Journal2020
Park, Jin Suk et al.Mechanical regulation of glycolysis via cytoskeleton architectureNature2020
Ergin, Volkan et al.Putative Coiled-Coil Domain-Dependent Autoinhibition and Alternative Splicing Determine SHTN1’s Actin-Binding ActivityJournal of Molecular Biology2020
Slater, Paula G. et al.XMAP215 promotes microtubule-F-actin interactions to regulate growth cone microtubules during axon guidance in Xenopus laevisJournal of cell science2019
Zhang, Shengnan et al.In-cell NMR study of Tau and MARK2 phosphorylated TauInternational Journal of Molecular Sciences2019
Figard, Lauren et al.Cofilin-Mediated Actin Stress Response Is Maladaptive in Heat-Stressed EmbryosCell Reports2019
Osório, Daniel S. et al.Crosslinking activity of non-muscle myosin II is not sufficient for embryonic cytokinesis in C. elegansDevelopment (Cambridge, England)2019
Antoku, Susumu et al.ERK1/2 Phosphorylation of FHOD Connects Signaling and Nuclear Positioning Alternations in Cardiac LaminopathyDevelopmental Cell2019
Silkworth, William T. et al.The neuron-specific formin Delphilin nucleates nonmuscle actin but does not enhance elongationMolecular biology of the cell2018
McIntosh, Betsy B. et al.Opposing Kinesin and Myosin-I Motors Drive Membrane Deformation and Tubulation along Engineered Cytoskeletal NetworksCurrent Biology2018
Olthoff, John T. et al.Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscleNature Communications2018
Raoux-Barbot, Dorothée et al.Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actinPLoS ONE2018
Balchin, David et al.Pathway of Actin Folding Directed by the Eukaryotic Chaperonin TRiCCell2018
Cervero, Pasquale et al.Lymphocyte-specific protein 1 regulates mechanosensory oscillation of podosomes and actin isoform-based actomyosin symmetry breakingNature Communications2018
Tsai, Feng Ching et al.Ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partnereLife2018
Cabrales Fontela, Yunior et al.Multivalent cross-linking of actin filaments and microtubules through the microtubule-associated protein TauNature Communications2017
Dräger, Nina M et al. Bin1 directly remodels actin dynamics through its BAR domain EMBO reports2017
Rondina, M. T. et al.Non-genomic activities of retinoic acid receptor alpha control actin cytoskeletal events in human plateletsJournal of Thrombosis and Haemostasis2016
Reeg, Sandra et al.The molecular chaperone Hsp70 promotes the proteolytic removal of oxidatively damaged proteins by the proteasomeFree Radical Biology and Medicine2016
Alqassim, Saif S. et al.Modulation of MICAL Monooxygenase Activity by its Calponin Homology Domain: Structural and Mechanistic InsightsScientific Reports2016
Neasta, Jeremie et al.Activation of the cAMP pathway induces RACK1-dependent binding of β-actin to BDNF promoterPLoS ONE2016
Belyy, Alexander et al.Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxinsNature Communications2016
Sobierajska, Katarzyna et al.Protein disulfide isomerase directly interacts with β-actin Cys374 and regulates cytoskeleton reorganizationThe Journal of biological chemistry2014
Lockett, Stephen et al.Quantitative analysis of F-actin redistribution in astrocytoma cells treated with candidate pharmaceuticalsCytometry Part A2014
Kremneva, Elena et al.Cofilin-2 controls actin filament length in muscle sarcomeresDevelopmental Cell2014
Lamothe, Betty et al.TAK1 is essential for osteoclast differentiation and is an important modulator of cell death by apoptosis and necroptosisMolecular and cellular biology2013
Gilmore, Jamie L. et al.AFM Investigation of the Organization of Actin Bundles Formed by Actin-Binding ProteinsJournal of Surface Engineered Materials and Advanced Technology2013
Marat, Andrea L. et al.Connecdenn 3/DENND1C binds actin linking Rab35 activation to the actin cytoskeletonMolecular Biology of the Cell2012
Gaidos, Gabriel et al.Structure and function analysis of the CMS/CIN85 protein family identifies actin-bundling properties and heterotypic-complex formationJournal of Cell Science2007
Gohla, Antje et al.Chronophin, a novel HAD-type serine protein phosphatase, regulates cofilin-dependent actin dynamicsNature cell biology2005
Posern, Guido et al.Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MALThe EMBO Journal2004
Searles, Charles D. et al.Actin cytoskeleton organization and posttranscriptional regulation of endothelial nitric oxide synthase during cell growthCirculation research2004
Roger, Benoit et al.MAP2c, but not tau, binds and bundles F-actin via its microtubule binding domain2004
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 vivoJournal of cell science2002
Kuriyama, Ryoko et al.CHO1, a mammalian kinesin-like protein, interacts with F-actin and is involved in the terminal phase of cytokinesisJournal of Cell Biology2002
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 activationMolecular biology of the cell2000
Vartiainen, Maria et al.Mouse A6/Twinfilin Is an Actin Monomer-Binding Protein That Localizes to the Regions of Rapid Actin DynamicsMolecular and Cellular Biology2000
Zhou, Daoguo et al.Role of the S. typhimurium Actin-Binding Protein SipA in Bacterial InternalizationScience1999

 Question 1: Do you have pyrene-labeled non-muscle actin for use in a polymerization assay?

Answer 1:  Pyrene-labeled non-muscle (beta gamma, 85:15) 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 tservice@cytoskeleton.com

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