Actin protein ( >99% pure): human platelet

Actin protein (>99% pure): human platelet
$0.00

Product Uses Include

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

Material
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.
 

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).

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
Park, Jin Suk et al.Mechanical regulation of glycolysis via cytoskeleton architectureNature2020ISSN 1476-4687
Ergin, Volkan et al.Putative Coiled-Coil Domain-Dependent Autoinhibition and Alternative Splicing Determine SHTN1’s Actin-Binding ActivityJournal of Molecular Biology2020ISSN 1089-8638
La, The Mon et al.Dynamin 1 is important for microtubule organization and stabilization in glomerular podocytesFASEB Journal2020ISSN 1530-6860
Antoku, Susumu et al.ERK1/2 Phosphorylation of FHOD Connects Signaling and Nuclear Positioning Alternations in Cardiac LaminopathyDevelopmental Cell2019ISSN 1878-1551
Osório, Daniel S. et al.Crosslinking activity of non-muscle myosin II is not sufficient for embryonic cytokinesis in C. elegansDevelopment (Cambridge, England)2019ISSN 1477-9129
Slater, Paula G. et al.XMAP215 promotes microtubule-F-actin interactions to regulate growth cone microtubules during axon guidance in Xenopus laevisJournal of cell science2019ISSN 1477--9137
Figard, Lauren et al.Cofilin-Mediated Actin Stress Response Is Maladaptive in Heat-Stressed EmbryosCell Reports2019ISSN 2211-1247
Zhang, Shengnan et al.In-cell NMR study of Tau and MARK2 phosphorylated TauInternational Journal of Molecular Sciences2019ISSN 1422-0067
McIntosh, Betsy B. et al.Opposing Kinesin and Myosin-I Motors Drive Membrane Deformation and Tubulation along Engineered Cytoskeletal NetworksCurrent Biology2018ISSN 0960-9822
Tsai, Feng Ching et al.Ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partnereLife2018ISSN 2050-084X
Silkworth, William T. et al.The neuron-specific formin Delphilin nucleates nonmuscle actin but does not enhance elongationMolecular biology of the cell2018ISSN 1939--4586
Cervero, Pasquale et al.Lymphocyte-specific protein 1 regulates mechanosensory oscillation of podosomes and actin isoform-based actomyosin symmetry breakingNature Communications2018ISSN 2041-1723
Balchin, David et al.Pathway of Actin Folding Directed by the Eukaryotic Chaperonin TRiCCell2018ISSN 1097-4172
Raoux-Barbot, Dorothée et al.Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actinPLoS ONE2018ISSN 1932-6203
Olthoff, John T. et al.Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscleNature Communications2018ISSN 2041-1723
Dräger, Nina M et al. Bin1 directly remodels actin dynamics through its BAR domain EMBO reports2017ISSN 1469--221X
Cabrales Fontela, Yunior et al.Multivalent cross-linking of actin filaments and microtubules through the microtubule-associated protein TauNature Communications2017ISSN 2041-1723
Belyy, Alexander et al.Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxinsNature Communications2016ISSN 2041-1723
Rondina, M. T. et al.Non-genomic activities of retinoic acid receptor alpha control actin cytoskeletal events in human plateletsJournal of Thrombosis and Haemostasis2016ISSN 1538-7836
Reeg, Sandra et al.The molecular chaperone Hsp70 promotes the proteolytic removal of oxidatively damaged proteins by the proteasomeFree Radical Biology and Medicine2016ISSN 1873-4596
Alqassim, Saif S. et al.Modulation of MICAL Monooxygenase Activity by its Calponin Homology Domain: Structural and Mechanistic InsightsScientific Reports2016ISSN 2045-2322
Neasta, Jeremie et al.Activation of the cAMP pathway induces RACK1-dependent binding of β-actin to BDNF promoterPLoS ONE2016ISSN 1932-6203
Lockett, Stephen et al.Quantitative analysis of F-actin redistribution in astrocytoma cells treated with candidate pharmaceuticalsCytometry Part A2014ISSN 1552-4930
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Sobierajska, Katarzyna et al.Protein disulfide isomerase directly interacts with β-actin Cys374 and regulates cytoskeleton reorganizationThe Journal of biological chemistry2014ISSN 1083--351X
Lamothe, Betty et al.TAK1 is essential for osteoclast differentiation and is an important modulator of cell death by apoptosis and necroptosisMolecular and cellular biology2013ISSN 1098--5549
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 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 tservice@cytoskeleton.com