Arp2/3 protein complex: porcine brain

Arp2/3 protein complex: porcine brain
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Product Uses Include

  • Positive control for in vitro actin polymerization stimualting compounds
  • Discovery of Arp2/3 activating or inhibiting compound
  • Characterization and discovery of Arp2/3 interacting proteins

Material
The Arp2/3 protein complex consists of 7 protein subunits, all present in approximately equal stoichiometry (see Figure 1). The Arp2/3 complex is a key regulator of actin filament nucleation and the protein subunits are highly evolutionarily conserved.  The protein complex has been purified from porcine brain and is supplied as a lyophilized powder.  When reconstituted with distilled water, the complex is in the following buffer: 20 mM Tris pH 7.5, 25 mM KCl, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM ATP, 1% dextran and 5% sucrose.  The molecular weight of the Arp2/3 complex is 224 kDa.

 

Note: RP01P from procine brain replaces RP01 from bovine brain. We have compared the activity of Arp2/3 complex from both sources and have found them to behave identically in actin polymerization assays (see Fig. 2)

 

Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gel. Arp2/3 protein complex is determined to be 90% pure (see Figure 1).

rp01p-fig1v2

Figure 1. Arp2/3 Protein Purity Determiniation: A 10 μg sample of Arp2/3 complex was separated by electro-phoresis in a 4-20% SDS-PAGE system and stained with Coomassie Blue. Protein quantita-tion was determined with the Precision RedTM Protein Assay Reagent (Cat. # ADV02). Mark12 molecular weight markers are from Life Technologies.

Biological Activity
RP01P was tested in an actin polymerization assay (Cat. # BK003). In conjunction with the VCA domain of WASP (Cat. # VCG03), an Arp2/3 activator, it was shown to stimulate actin polymerization by 20-fold compared to the control without RP01. This indicates Arp 2/3 specificity for actin polymerization induction (Figure 2).

rp01p-fig2v2

Figure 2. Actin Polymerization Assay with Arp2/3 and VCA domain proteins: Actin polymerization was carried out as described in the method; All reactions contain 0.8 μM actin. Reactions containing Arp2/3 or VCA (data not shown) show little or no enhancement of actin nucleation. Actin alone also shows low polymerization com-petence under these conditions (Actin alone reactions). In the presence of the VCA domain however, Arp2/3 results in an en-hancement of actin nucleation (Note the steep nucleation phase of polymerization). Actin polymerization is measured in arbitrary fluorescent units over time. In our tests porcine derived Arp2/3(wells E & F) behaved identically to Arp2/3 derived from bovine sources (wells G & H).

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

AuthorTitleJournalYearArticle Link
Sakamoto, Ryota et al.F-actin architecture determines the conversion of chemical energy into mechanical workNature Communications 2024ISSN 2041--1723
Nakamura, Hideki et al.ActuAtor, a Listeria-inspired molecular tool for physical manipulation of intracellular organizations through de novo actin polymerizationCell reports2023ISSN 2211--1247
Li, Dong et al.GxcM-Fbp17/RacC-WASP signaling regulates polarized cortex assembly in migrating cells via Arp2/3Journal of Cell Biology2023ISSN 1540-8140
Cao, Muqing et al.An actin filament branching surveillance system regulates cell cycle progression, cytokinesis and primary ciliogenesisNature Communications 2023 14:12023ISSN 2041--1723
Bogucka-Janczi, Katarzyna et al.ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization.eLife2023ISSN 2050--084X
Bashirzadeh, Yashar et al.Encapsulated actomyosin patterns drive cell-like membrane shape changesiScience2022
Bock, Fabian et al.Rac1 promotes kidney collecting duct integrity by limiting actomyosin activityJournal of Cell Biology2021ISSN 1540-8140
Lin, Shan Shan et al.Dynamin-2 Regulates Postsynaptic Cytoskeleton Organization and Neuromuscular Junction DevelopmentCell Reports2020ISSN 2211-1247
Sonal et al.Myosin-II activity generates a dynamic steady state with continuous actin turnover in a minimal actin cortexJournal of Cell Science2019ISSN 1477-9137
Galvan, Daniel L. et al.Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic miceJournal of Clinical Investigation2019ISSN 1558-8238
Schaffer, Ashleigh E. et al.Biallelic loss of human CTNNA2, encoding αN-catenin, leads to ARP2/3 complex overactivity and disordered cortical neuronal migrationNature Genetics2018ISSN 1546-1718
Zeng, Menglong et al.Reconstituted Postsynaptic Density as a Molecular Platform for Understanding Synapse Formation and PlasticityCell2018ISSN 1097-4172
Tu, Ye et al.EFhd2/swiprosin-1 regulates LPS-induced macrophage recruitment via enhancing actin polymerization and cell migrationInternational Immunopharmacology2018ISSN 1878-1705
Mikhaylova, Marina et al.Caldendrin Directly Couples Postsynaptic Calcium Signals to Actin Remodeling in Dendritic SpinesNeuron2018ISSN 1097-4199
Almeida-Souza, Leonardo et al.A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated PitsCell2018ISSN 1097-4172
Bertier, Laurence et al.Inhibitory cortactin nanobodies delineate the role of NTA- and SH3-domain-specific functions during invadopodium formation and Cancer cell invasionFASEB Journal2017ISSN 1530-6860
Zhao, Miao et al.Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerizationOncotarget2017ISSN 1949-2553
Roybal, Kole T. et al.Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamicsScience Signaling2016ISSN 1937-9145
Chellaiah, Meenakshi A. et al.Phosphorylation of a Wiscott-Aldrich syndrome protein-associated signal complex is critical in osteoclast bone resorptionJournal of Biological Chemistry2007ISSN 0021-9258
Van Der Gucht, Jasper et al.Stress release drives symmetry breaking for actin-based movementProceedings of the National Academy of Sciences of the United States of America2005ISSN 0027-8424
Leng, Yan et al.Abelson-interactor-1 promotes WAVE2 membrane translocation and Abelson-mediated tyrosine phosphory lation required for WAVE2 activationProceedings of the National Academy of Sciences of the United States of America2005ISSN 0027-8424


Note: RP01P from procine brain replaces RP01 from bovine brain. We have compared the activity of Arp2/3 complex from both sources and have found them to behave identically in actin polymerization assays.  The assays below reference the Arp2/3 protein complex: bovine brain (Cat. RP01).  Please inquire to tservice@cytoskeleton.com for the latest papers citing this product.

Leng, Y., Zhang, J., Badour, K., Arpaia, E., Freeman, S., Cheung, P., Siu, M. and Siminovitch, K. (2005). Abelson-interactor-1 promotes WAVE2 membrane translocation and Abelson-mediated tyrosine phosphorylation required for WAVE2 activation. Proc. Natl. Acad. Sci. U. S. A. 102, 1098-1103.

van der Gucht, J., Paluch, E., Plastino, J. and Sykes, C. (2005). Stress release drives symmetry breaking for actin-based movement. Proc. Natl. Acad. Sci. U. S. A. 102, 7847-7852.

Question 1:  What is the best way to store Arp2/3 (Cat. # RP01P)?

Answer 1:  Store the lyophilized Arp2/3 protein desiccated (<10% humidity) at 4°C where it is stable for 6 months.  Lyophilized protein can also be stored desiccated at -70°C where it will be stable for 6 months.  However, at -70°C the rubber seal in the lid of the tube could crack and allow in moisture.  Therefore we recommend storing at 4°C.  If stored at -70°C, it is imperative to include desiccant with the lyophilized protein if this storage condition is utilized.    Resuspend the protein complex to 5 mg/ml with 10 μl of cold Milli-Q water.  When resuspended, the complex is in the following buffer: 20 mM Tris pH 7.5, 25 mM KCl, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM ATP, 1.0% (v/v) dextran and 5% (v/v) sucrose.  The protein should then be aliquoted into experiment-sized amounts, snap frozen in liquid nitrogen and stored at -70°C where it is stable for 6 months.  Further dilution of Arp2/3 should be made in the following buffer: 20 mM Tris pH 7.5, 25 mM KCl, 1 mM MgCl2 and 1 mM DTT (Note: add DTT to the buffer immediately prior to use).  NOTE: It is very important to snap freeze the Arp2/3 protein in liquid nitrogen as other methods of freezing will result in significantly reduced activity.  Defrost rapidly by placing in a room temperature water bath for 1 min.  Avoid repeated freeze/thaw cycles.

 

Question 2:  Is the Arp2/3 (Cat. # RP01P) compatible with the actin polymerization kit (Cat. # BK003)?

Answer 2:  Yes, Arp2/3 works very well with the actin polyermization kit (Cat. # BK003) to study the effects of this actin binding protein on actin polymerization.  Please see the Arp2/3 datasheet (Cat. # RP01P) for additional information.  The Arp2/3 complex is able to induce the branched polymerization of actin filaments in vitro at a molar ratio of 1:200 (Arp2/3:actin).  This stimulation is observable in an in vitro polymerization assay; however, the stimulation from Arp2/3 alone is very low under typical polymerization conditions.  In the presence of N-WASP protein (or the VCA domain of N-WASP, Cat. # VCG03), the nucleating activity of Arp2/3 is greatly enhanced.  In the polymerization assay described in the Arp2/3 datasheet, pyrene actin (Cat. # AP05 or BK003) is present at a final concentration of 0.8 μM, the Arp2/3 complex is at 10 nM and the VCA domain (Cat. # VCG03) is at 400 nM.

 

If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com.