Antibodies

Highly characterized antibodies with validated applications

Each antibody is developed in-house and rigorously tested for specificity and sensitivity in western blot, immunofluorescence, immunoprecipitation, and ELISA.  For more information, click on the appropriate group of antigens below.

 

  • Validated for Multiple Applications
  • Developed In-House
  • Lot-to-Lot Consistency
  • High Specificity and Sensitivity
Antibody-CatImg

At Cytoskeleton, we understand that marketplace antibodies can be dubious and perform inconsistently.  Conversely, our antibodies go through a rigorous validation process to give scientists the best picture of an antibody’s performance in different situations.  An example of typical validation tests that are performed are described below.

1) Western blot

Western blot gives great information about specificity (the ability to detect only the antigen), and some information about the affinity. If the antibody can detect one antigen band in a lane which is loaded with 50 µg of extract then it is considered specific. If it can detect 1 ng (50 femto-moles) of antigen using chemiluminescence detection it is considered high affinity.

arc03-wb

Figure 1.  Western blot analysis of anti-Rac1 antibody (Cat. # ARC03). The detection of 25 ng His-Rac1 (lane 1), His-Cdc42 (lane 2), 50 µg of platelet extract (lane 3), His-Rac2 (lane 4) and His-Rac3 (lane 5).  ARC03 does not cross-react with Rac2, 3, or Cdc42. The blot was probed with a 1 µg/ml (1:500) dilution of ARC03, 30 second exposure time.

2) Immunofluorescence in situ

Immunofluorescence in situ (IF) and its counterparts immunohistochemistry (IH), fluorescence activated cell sorting (FACS) and high content screening (HCS) are performed by staining live or fixed cells or tissues with a fluorescent or colorimetric marker. The importance of this technique to characterize antibodies is to confirm that the reagent can detect the antigen in its native environment. An antibody may detect the native conformation (e.g., with paraformaldehyde fixation) or denatured antigen (e.g., with methanol fixation) and hence these aspects must be presented in the datasheet. Another consideration is that the antigen may be masked by another molecule e.g., protein or DNA, and hence may not work in this application unless a disrupting agent (e.g., methanol, detergent, or chaotropic agent) is added first. A pre-requisite of this technique is to be sure the western blot is giving just one band in a lane containing cell extract.

ATN02-S

Figure 2. Immunofluorescence images of mouse Swiss 3T3 cells stained with anti-tubulin  polyclonal antibody (Cat. # ATN02). Swiss 3T3 cells were grown to semi-confluency and fixed with methanol. Immunofluorescence staining using 2.5 µg/ml (1:200 dilution) ATN02 antibody is shown (red). The primary antibody was detected with a 1:500 dilution of anti-sheep rhodamine conjugated antibody. Photograph was taken with a 100X objective lens.

3) ELISA

ELISA is a quantitative technique to determine the amount of antigen in a sample. The technique is important in determining the affinity of an antibody to an antigen. Usually high-affinity reagents have Kd = 10-9 M or higher.

ARH03_Kd_measurement

Figure 3:  Each well of an ELISA plate was coated with 2 ng of RH01, then blocked with dry milk and probed with sequential dilutions of ARH03. Bound antibody was detected with HRP-secondary anti-mouse antibody and signals developed with OPD reagent. The OD490nm signals were plotted on the Y-axis to create a binding curve. The Kd was calculated from the 50% full scale signal X-axis intercept

4) Immunoprecipitation

Immunoprecipitation (Ippt) is a useful technique to determine binding partners in an extract. It is particularly useful in signal transduction research where regulated binding partners may differentially occur in the pellet sample when activated or de-activated. The antibody is usually bound to a bead and used as an affinity matrix to pullout the antigen and any associated proteins.

anti-phosphotyrosine-ip_2

Figure 4. Immunoprecipitation of pervanadate-treated lysates from NIH 3T3 cells using APY03.  NIH 3T3 cells were either treated (+) or untreated (-) with H2O2/orthovanadate (100 µM for 10min). Cell lysate was prepared in RIPA buffer and 200 µg of lysate per reaction was used forimmunoprecipitation of tyrosine-phosphorylated proteins. APY03 was first bound to protein G-beads and then incubated with cell lysate. For bead-only control, cell lysate was incubated with protein G-beads without APY03. Western blots of immunoprecipitated proteins were developed using APY03 at 1:500 dilution and CleanBlot (Thermo Scientific, #21230) as secondary antibody. No IP input lysate represents the signal from 5% of H2O2/orthovanadate treated or untreated NIH 3T3 lysate. As shown in Figure 2, APY03 was able to enrich a wide range of tyrosine-phosphorylated proteins from NIH 3T3 cells treated with H2O2-activated orthovanadate. No signal was detected with protein G-bead control without APY03. 

Cytoskeleton's antibody products have been cited hundreds of times over the past two decades. More individual product citations listed on each product page.


AuthorTitleJournalYearArticle Link
Kamata, Tamihiro et al.Statins mediate anti- and pro-tumourigenic functions by remodelling the tumour microenvironmentDMM Disease Models and Mechanisms2022ISSN 1754-8411
Barbera, Stefano et al.The C-type lectin CD93 controls endothelial cell migration via activation of the Rho family of small GTPasesMatrix Biology2021ISSN 1569-1802
Howden, Jake D. et al.α2β1 integrins spatially restrict Cdc42 activity to stabilise adherens junctionsBMC biology2021ISSN 1741-7007
Li, Jingxuan et al.Eva1a ameliorates atherosclerosis by promoting re-endothelialization of injured arteries via Rac1/Cdc42/Arpc1bCardiovascular Research2021ISSN 1755-3245
Jayabal, Panneerselvam et al.NELL2-cdc42 signaling regulates BAF complexes and Ewing sarcoma cell growthCell Reports2021ISSN 2211-1247
Hosseini, Kamran et al.EMT-Induced Cell-Mechanical Changes Enhance Mitotic Rounding StrengthAdvanced Science2020ISSN 2198-3844
Gorisse, Laetitia et al.Ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the Rho GTPase CDC42Journal of Biological Chemistry2020ISSN 1083-351X
Gu, Jiawen et al.Rho-GEF trio regulates osteoclast differentiation and function by Rac1/Cdc42Experimental Cell Research2020ISSN 1090-2422
Oni, Tobiloba E. et al.SOAT1 promotes mevalonate pathway dependency in pancreatic cancerJournal of Experimental Medicine2020ISSN 1540-9538
Vestre, Katharina et al.Rab6 regulates cell migration and invasion by recruiting Cdc42 and modulating its activityCellular and Molecular Life Sciences2019ISSN 1420-9071
Singh, Rajesh K. et al.Dynamic Actin Reorganization and Vav/Cdc42-Dependent Actin Polymerization Promote Macrophage Aggregated LDL (Low-Density Lipoprotein) Uptake and CatabolismArteriosclerosis, Thrombosis, and Vascular Biology2019ISSN 1524-4636
Dupraz, Sebastian et al.RhoA Controls Axon Extension Independent of Specification in the Developing BrainCurrent Biology2019ISSN 0960-9822
Huang, Yuxing et al.Arp2/3-branched actin maintains an active pool of GTP-RhoA and controls RhoA abundanceCells2019ISSN 2073-4409
MacKeil, Jodi L. et al.Phosphodiesterase 3B (PDE3B) antagonizes the anti-angiogenic actions of PKA in human and murine endothelial cellsCellular Signalling2019ISSN 1873-3913
Li, Cao et al.Regulation of staphylococcus aureus infection of macrophages by CD44, reactive oxygen species, and acid sphingomyelinaseAntioxidants and Redox Signaling2018ISSN 1557-7716
Stypulkowski, Ewa et al.The depalmitoylase APT1 directs the asymmetric partitioning of Notch and Wnt signaling during cell divisionScience Signaling2018ISSN 1937-9145
Kawasaki, Natsumi et al.TUFT1 interacts with RABGAP1 and regulates mTORC1 signalingCell Discovery2018ISSN 2056-5968
Ghézali, Grégory et al.Connexin 30 controls astroglial polarization during postnatal brain developmentDevelopment (Cambridge)2018ISSN 1477-9129
Vodicska, Barbara et al.MISP regulates the IQGAP1/Cdc42 complex to collectively orchestrate spindle orientation and mitotic progressionScientific Reports2018ISSN 2045-2322
Wu, Nan et al.RCC2 over-expression in tumor cells alters apoptosis and drug sensitivity by regulating Rac1 activationBMC Cancer2018ISSN 1471-2407
Lam, Jonathan G.T. et al.Host cell perforation by listeriolysin O (LLO) activates a Ca2+-dependent cPKC/Rac1/Arp2/3 signaling pathway that promotes Listeria monocytogenes internalization independently of membrane resealingMolecular Biology of the Cell2018ISSN 1939-4586
Liu, Xiaolei et al.Rasip1 controls lymphatic vessel lumen maintenance by regulating endothelial cell junctionsDevelopment (Cambridge)2018ISSN 1477-9129
Peretti, Amanda S. et al.The R-Enantiomer of Ketorolac Delays Mammary Tumor Development in Mouse Mammary Tumor Virus-Polyoma Middle T Antigen (MMTV-PyMT) MiceAmerican Journal of Pathology2018ISSN 1525-2191
Dudvarski Stanković, Nevenka et al. EGFL7 enhances surface expression of integrin α 5 β 1 to promote angiogenesis in malignant brain tumors EMBO Molecular Medicine2018ISSN 1757--4676
Saito, Masaki et al.Tctex‐1 controls ciliary resorption by regulating branched actin polymerization and endocytosisEMBO reports2017ISSN 1469--221X
Bucka, Kenneth B. et al.Local Arp2/3-dependent actin assembly modulates applied traction force during apCAM adhesion site maturationMolecular Biology of the Cell2017ISSN 1939-4586
Chang, Ting Ya et al.Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machineryeLife2017ISSN 2050-084X
Takeuchi, Hiroki et al.Intracellular periodontal pathogen exploits recycling pathway to exit from infected cellsCellular Microbiology2016ISSN 1462-5822
Seidelin, Jakob Benedict et al.Cellular inhibitor of apoptosis protein 2 controls human colonic epithelial restitution, migration, and Rac1 activationAmerican Journal of Physiology - Gastrointestinal and Liver Physiology2015ISSN 1522-1547
Thomas, Audrey et al.Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T CellsJournal of Virology2015ISSN 0022--538X
Liu, Chunqiao et al.Null and hypomorph Prickle1 alleles in mice phenocopy human Robinow syndrome and disrupt signaling downstream of Wnt5aBiology Open2014ISSN 2046-6390
David, Muriel D. et al.The RhoGAP ARHGAP19 controls cytokinesis and chromosome segregation in T lymphocytesJournal of Cell Science2014ISSN 0021-9533
Valtcheva, Nadejda et al.The orphan adhesion G protein-coupled receptor GPR97 regulates migration of lymphatic endothelial cells via the small GTPases RhoA and Cdc42Journal of Biological Chemistry2013ISSN 0021-9258
Barrio, Laura et al.TLR4 Signaling Shapes B Cell Dynamics via MyD88-Dependent Pathways and Rac GTPasesThe Journal of Immunology2013ISSN 0022--1767
Steele, Brian M. et al.WNT-3a modulates platelet function by regulating small GTPase activityFEBS Letters2012ISSN 0014-5793
Mercer, Jason et al.Vaccinia virus strains use distinct forms of macropinocytosis for host-cell entryProceedings of the National Academy of Sciences of the United States of America2010ISSN 0027-8424
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