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.
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.
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.
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.
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.
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.
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.
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
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.
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.
|Kamata, Tamihiro et al.||Statins mediate anti- and pro-tumourigenic functions by remodelling the tumour microenvironment||DMM Disease Models and Mechanisms||2022||ISSN 1754-8411|
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|Howden, Jake D. et al.||α2β1 integrins spatially restrict Cdc42 activity to stabilise adherens junctions||BMC biology||2021||ISSN 1741-7007|
|Li, Jingxuan et al.||Eva1a ameliorates atherosclerosis by promoting re-endothelialization of injured arteries via Rac1/Cdc42/Arpc1b||Cardiovascular Research||2021||ISSN 1755-3245|
|Jayabal, Panneerselvam et al.||NELL2-cdc42 signaling regulates BAF complexes and Ewing sarcoma cell growth||Cell Reports||2021||ISSN 2211-1247|
|Hosseini, Kamran et al.||EMT-Induced Cell-Mechanical Changes Enhance Mitotic Rounding Strength||Advanced Science||2020||ISSN 2198-3844|
|Gorisse, Laetitia et al.||Ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the Rho GTPase CDC42||Journal of Biological Chemistry||2020||ISSN 1083-351X|
|Gu, Jiawen et al.||Rho-GEF trio regulates osteoclast differentiation and function by Rac1/Cdc42||Experimental Cell Research||2020||ISSN 1090-2422|
|Oni, Tobiloba E. et al.||SOAT1 promotes mevalonate pathway dependency in pancreatic cancer||Journal of Experimental Medicine||2020||ISSN 1540-9538|
|Vestre, Katharina et al.||Rab6 regulates cell migration and invasion by recruiting Cdc42 and modulating its activity||Cellular and Molecular Life Sciences||2019||ISSN 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 Catabolism||Arteriosclerosis, Thrombosis, and Vascular Biology||2019||ISSN 1524-4636|
|Dupraz, Sebastian et al.||RhoA Controls Axon Extension Independent of Specification in the Developing Brain||Current Biology||2019||ISSN 0960-9822|
|Huang, Yuxing et al.||Arp2/3-branched actin maintains an active pool of GTP-RhoA and controls RhoA abundance||Cells||2019||ISSN 2073-4409|
|MacKeil, Jodi L. et al.||Phosphodiesterase 3B (PDE3B) antagonizes the anti-angiogenic actions of PKA in human and murine endothelial cells||Cellular Signalling||2019||ISSN 1873-3913|
|Li, Cao et al.||Regulation of staphylococcus aureus infection of macrophages by CD44, reactive oxygen species, and acid sphingomyelinase||Antioxidants and Redox Signaling||2018||ISSN 1557-7716|
|Stypulkowski, Ewa et al.||The depalmitoylase APT1 directs the asymmetric partitioning of Notch and Wnt signaling during cell division||Science Signaling||2018||ISSN 1937-9145|
|Kawasaki, Natsumi et al.||TUFT1 interacts with RABGAP1 and regulates mTORC1 signaling||Cell Discovery||2018||ISSN 2056-5968|
|Ghézali, Grégory et al.||Connexin 30 controls astroglial polarization during postnatal brain development||Development (Cambridge)||2018||ISSN 1477-9129|
|Vodicska, Barbara et al.||MISP regulates the IQGAP1/Cdc42 complex to collectively orchestrate spindle orientation and mitotic progression||Scientific Reports||2018||ISSN 2045-2322|
|Wu, Nan et al.||RCC2 over-expression in tumor cells alters apoptosis and drug sensitivity by regulating Rac1 activation||BMC Cancer||2018||ISSN 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 resealing||Molecular Biology of the Cell||2018||ISSN 1939-4586|
|Liu, Xiaolei et al.||Rasip1 controls lymphatic vessel lumen maintenance by regulating endothelial cell junctions||Development (Cambridge)||2018||ISSN 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) Mice||American Journal of Pathology||2018||ISSN 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 Medicine||2018||ISSN 1757--4676|
|Saito, Masaki et al.||Tctex‐1 controls ciliary resorption by regulating branched actin polymerization and endocytosis||EMBO reports||2017||ISSN 1469--221X|
|Bucka, Kenneth B. et al.||Local Arp2/3-dependent actin assembly modulates applied traction force during apCAM adhesion site maturation||Molecular Biology of the Cell||2017||ISSN 1939-4586|
|Chang, Ting Ya et al.||Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery||eLife||2017||ISSN 2050-084X|
|Takeuchi, Hiroki et al.||Intracellular periodontal pathogen exploits recycling pathway to exit from infected cells||Cellular Microbiology||2016||ISSN 1462-5822|
|Seidelin, Jakob Benedict et al.||Cellular inhibitor of apoptosis protein 2 controls human colonic epithelial restitution, migration, and Rac1 activation||American Journal of Physiology - Gastrointestinal and Liver Physiology||2015||ISSN 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 Cells||Journal of Virology||2015||ISSN 0022--538X|
|Liu, Chunqiao et al.||Null and hypomorph Prickle1 alleles in mice phenocopy human Robinow syndrome and disrupt signaling downstream of Wnt5a||Biology Open||2014||ISSN 2046-6390|
|David, Muriel D. et al.||The RhoGAP ARHGAP19 controls cytokinesis and chromosome segregation in T lymphocytes||Journal of Cell Science||2014||ISSN 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 Cdc42||Journal of Biological Chemistry||2013||ISSN 0021-9258|
|Barrio, Laura et al.||TLR4 Signaling Shapes B Cell Dynamics via MyD88-Dependent Pathways and Rac GTPases||The Journal of Immunology||2013||ISSN 0022--1767|
|Steele, Brian M. et al.||WNT-3a modulates platelet function by regulating small GTPase activity||FEBS Letters||2012||ISSN 0014-5793|
|Mercer, Jason et al.||Vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry||Proceedings of the National Academy of Sciences of the United States of America||2010||ISSN 0027-8424|