Immunofluorescence analysis of Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate was performed using A549 cells stained with alpha Tubulin (YL1/2) Rat Monoclonal Antibody (Product # MA1-80017). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, blocked with 1% BSA for 1 hour and labeled with 2µg/ml Rat primary antibody for 3 hours at room temperature. Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate (Product # A11007) was used at a concentration of 4µg/ml in phosphate buffered saline containing 0.2 % BSA for 45 minutes at room temperature, for detection of alpha Tubulin in the cytoplasm (Panel a: red). Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (Product # S36938). F-actin was stained with Alexa Fluor® 488 Phalloidin (Product # A12379), 1:300) (Panel c: green). Panel d represents the composite image. No nonspecific staining was observed with the secondary antibody alone (panel f), or with an isotype control (panel e). The images were captured at 60X magnification.
|Tested species reactivity||Rat|
|Published species reactivity||Not Applicable|
|Host / Isotype||Goat / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 594|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Cross Adsorption||Against mouse IgG, mouse serum and human serum prior to conjugation|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:500|
|Immunocytochemistry (ICC)||4 µg/ml|
|Immunofluorescence (IF)||4 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
To minimize cross-reactivity, these goat anti-rat IgG (H+L) whole secondary antibodies have been affinity purified and cross-adsorbed against mouse IgG, mouse serum, and human serum prior to conjugation. Cross-adsorption or pre-adsorption is a purification step to increase specificity of the antibody resulting in higher sensitivity and less background staining. The secondary antibody solution is passed through a column matrix containing immobilized serum proteins from potentially cross-reactive species. Only the nonspecific-binding secondary antibodies are captured in the column, and the highly specific secondaries flow through. The benefits of this extra step are apparent in multiplexing/multicolor-staining experiments (e.g., flow cytometry) where there is potential cross-reactivity with other primary antibodies or in tissue/cell fluorescent staining experiments where there are may be the presence of endogenous immunoglobulins.
Alexa Fluor dyes are among the most trusted fluorescent dyes available today. Invitrogen™ Alexa Fluor 594 dye is a bright, red-fluorescent dye with excitation ideally suited to the 594 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 594 dye is pH-insensitive over a wide molar range. Probes with high fluorescence quantum yield and high photostability allow detection of low-abundance biological structures with great sensitivity. Alexa Fluor 594 dye molecules can be attached to proteins at high molar ratios without significant self-quenching, enabling brighter conjugates and more sensitive detection. The degree of labeling for each conjugate is typically 2-8 fluorophore molecules per IgG molecule; the exact degree of labeling is indicated on the certificate of analysis for each product lot.
Using conjugate solutions: Centrifuge the protein conjugate solution briefly in a microcentrifuge before use; add only the supernatant to the experiment. This step will help eliminate any protein aggregates that may have formed during storage, thereby reducing nonspecific background staining. Because staining protocols vary with application, the appropriate dilution of antibody should be determined empirically. For the fluorophore-labeled antibodies a final concentration of 1-10 µg/mL should be satisfactory for most immunohistochemistry and flow cytometry applications.
We offer an extensive line of Invitrogen™ secondary antibody conjugates with well-characterized specificity and labeled with a wide selection of premium fluorescent dyes, including Invitrogen™ Alexa Fluor™ fluorescent dyes. Fluorescent secondary antibody conjugates are useful in the detection, sorting, or purification of its specified target and ideal for fluorescence microscopy and confocal laser scanning microscopy, flow cytometry, and fluorescent western detection. The breadth of fluorescent markers we offer allows our reagents to be tailored to almost any fluorescent detection system.
Secondary antibodies may be provided in three formats: whole IgG, divalent F(ab')2 fragments, and monovalent Fab fragments. Because of the high degree of conservation in the structure of many immunoglobulin domains, most class-specific secondary antibodies must be affinity-purified and cross-adsorbed to achieve minimal cross-reaction with other immunoglobulins.
Our secondary antibody conjugates are most commonly prepared by immunizing the host animal with a pooled population of immunoglobulins from the target species and can be further purified and modified (e.g., immunoaffinity chromatography, antibody fragmentation, label conjugation, etc.) to generate highly specific reagents. In the first round of purification, whole immunoglobulins binding to the immunizing antibody are recovered and mainly consist of the ~150-kDa IgG class. Further purification, for example, with Protein A or G, removes all unwanted immunoglobulin classes except the affinity-purified antibodies that react with the target-specific immunoglobulin heavy and/or light chains.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Not Applicable||Not Cited||Bacterial recognition by TLR7 in the lysosomes of conventional dendritic cells.||Mancuso G,Gambuzza M,Midiri A,Biondo C,Papasergi S,Akira S,Teti G,Beninati C||Nature immunology (10:587)||2009|
|Not Applicable||Not Cited||Involvement of mitochondrial signaling pathways in the mechanism of Fas-mediated apoptosis after spinal cord injury.||Yu WR,Liu T,Fehlings TK,Fehlings MG||The European journal of neuroscience (29:114)||2009|
|Not Applicable||Not Cited||In vivo tumor cell targeting with "click" nanoparticles.||von Maltzahn G,Ren Y,Park JH,Min DH,Kotamraju VR,Jayakumar J,Fogal V,Sailor MJ,Ruoslahti E,Bhatia SN||Bioconjugate chemistry (19:1570)||2008|
|Not Applicable||Not Cited||Imaging epidermal growth factor receptor expression in vivo: pharmacokinetic and biodistribution characterization of a bioconjugated quantum dot nanoprobe.||Diagaradjane P,Orenstein-Cardona JM,Colón-Casasnovas NE,Deorukhkar A,Shentu S,Kuno N,Schwartz DL,Gelovani JG,Krishnan S||Clinical cancer research : an official journal of the American Association for Cancer Research (14:731)||2008|
|Not Applicable||Not Cited||In vivo negative selection screen identifies genes required for Francisella virulence.||Weiss DS,Brotcke A,Henry T,Margolis JJ,Chan K,Monack DM||Proceedings of the National Academy of Sciences of the United States of America (104:6037)||2007|
|Not Applicable||Not Cited||The contribution of bone marrow-derived cells to the development of renal interstitial fibrosis.||Li J,Deane JA,Campanale NV,Bertram JF,Ricardo SD||Stem cells (Dayton, Ohio) (25:697)||2007|
|Not Applicable||Not Cited||The tetraspanin CD9 mediates lateral association of MHC class II molecules on the dendritic cell surface.||Unternaehrer JJ,Chow A,Pypaert M,Inaba K,Mellman I||Proceedings of the National Academy of Sciences of the United States of America (104:234)||2007|
|Not Applicable||Not Cited||The P2Y12 receptor regulates microglial activation by extracellular nucleotides.||Haynes SE,Hollopeter G,Yang G,Kurpius D,Dailey ME,Gan WB,Julius D||Nature neuroscience (9:1512)||2006|
|Not Applicable||Not Cited||Neocortical neurogenesis in humans is restricted to development.||Bhardwaj RD,Curtis MA,Spalding KL,Buchholz BA,Fink D,Björk-Eriksson T,Nordborg C,Gage FH,Druid H,Eriksson PS,Frisén J||Proceedings of the National Academy of Sciences of the United States of America (103:12564)||2006|
|Not Applicable||Not Cited||The EphB4 receptor suppresses breast cancer cell tumorigenicity through an Abl-Crk pathway.||Noren NK,Foos G,Hauser CA,Pasquale EB||Nature cell biology (8:815)||2006|
|Not Applicable||Not Cited||Imaging of CNS myelin by positron-emission tomography.||Stankoff B,Wang Y,Bottlaender M,Aigrot MS,Dolle F,Wu C,Feinstein D,Huang GF,Semah F,Mathis CA,Klunk W,Gould RM,Lubetzki C,Zalc B||Proceedings of the National Academy of Sciences of the United States of America (103:9304)||2006|
|Not Applicable||Not Cited||The chromatoid body of male germ cells: similarity with processing bodies and presence of Dicer and microRNA pathway components.||Kotaja N,Bhattacharyya SN,Jaskiewicz L,Kimmins S,Parvinen M,Filipowicz W,Sassone-Corsi P||Proceedings of the National Academy of Sciences of the United States of America (103:2647)||2006|
|Not Applicable||Not Cited||Extracellular application of nicotinic acid adenine dinucleotide phosphate induces Ca2+ signaling in astrocytes in situ.||Heidemann AC,Schipke CG,Kettenmann H||The Journal of biological chemistry (280:35630)||2005|
|Not Applicable||Not Cited||Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/LAMP-2 double-deficient fibroblasts.||Eskelinen EL,Schmidt CK,Neu S,Willenborg M,Fuertes G,Salvador N,Tanaka Y,Lüllmann-Rauch R,Hartmann D,Heeren J,von Figura K,Knecht E,Saftig P||Molecular biology of the cell (15:3132)||2004|
|Not Applicable||Not Cited||Olfactory horizontal basal cells demonstrate a conserved multipotent progenitor phenotype.||Carter LA,MacDonald JL,Roskams AJ||The Journal of neuroscience : the official journal of the Society for Neuroscience (24:5670)||2004|
|Not Applicable||Not Cited||Regulation of phagosome maturation by signals from toll-like receptors.||Blander JM,Medzhitov R||Science (New York, N.Y.) (304:1014)||2004|
|Not Applicable||Not Cited||Differential expression of heparan sulfate domains in rat spleen.||ten Dam GB,Hafmans T,Veerkamp JH,van Kuppevelt TH||The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society (51:727)||2003|
|Not Applicable||Not Cited||Control of axon elongation via an SDF-1alpha/Rho/mDia pathway in cultured cerebellar granule neurons.||Arakawa Y,Bito H,Furuyashiki T,Tsuji T,Takemoto-Kimura S,Kimura K,Nozaki K,Hashimoto N,Narumiya S||The Journal of cell biology (161:381)||2003|
|Not Applicable||Not Cited||Sumoylation of the progesterone receptor and of the steroid receptor coactivator SRC-1.||Chauchereau A,Amazit L,Quesne M,Guiochon-Mantel A,Milgrom E||The Journal of biological chemistry (278:12335)||2003|
|Not Applicable||Not Cited||Migration of nerve growth cones requires detergent-resistant membranes in a spatially defined and substrate-dependent manner.||Nakai Y,Kamiguchi H||The Journal of cell biology (159:1097)||2002|
|Not Applicable||Not Cited||Regulation of reticuloendothelial iron transporter MTP1 (Slc11a3) by inflammation.||Yang F,Liu XB,Quinones M,Melby PC,Ghio A,Haile DJ||The Journal of biological chemistry (277:39786)||2002|
|Not Applicable||Not Cited||Proteinase suppression by E-cadherin-mediated cell-cell attachment in premalignant oral keratinocytes.||Munshi HG,Ghosh S,Mukhopadhyay S,Wu YI,Sen R,Green KJ,Stack MS||The Journal of biological chemistry (277:38159)||2002|
|Not Applicable||Not Cited||Identification and characterization of RPK118, a novel sphingosine kinase-1-binding protein.||Hayashi S,Okada T,Igarashi N,Fujita T,Jahangeer S,Nakamura S||The Journal of biological chemistry (277:33319)||2002|
|Not Applicable||Not Cited||Distinct intracellular calcium transients in neurites and somata integrate neuronal signals.||Johenning FW,Zochowski M,Conway SJ,Holmes AB,Koulen P,Ehrlich BE||The Journal of neuroscience : the official journal of the Society for Neuroscience (22:5344)||2002|
|Not Applicable||Not Cited||In vivo analysis of human multidrug resistance protein 1 (MRP1) activity using transient expression of fluorescently tagged MRP1.||Rajagopal A,Pant AC,Simon SM,Chen Y||Cancer research (62:391)||2002|
|Not Applicable||Not Cited||EphB/syndecan-2 signaling in dendritic spine morphogenesis.||Ethell IM,Irie F,Kalo MS,Couchman JR,Pasquale EB,Yamaguchi Y||Neuron (31:1001)||2001|
|Not Applicable||Not Cited||The intracellular localization of the mineralocorticoid receptor is regulated by 11beta-hydroxysteroid dehydrogenase type 2.||Odermatt A,Arnold P,Frey FJ||The Journal of biological chemistry (276:28484)||2001|
|Not Applicable||Not Cited||Matrix-dependent proteolysis of surface transglutaminase by membrane-type metalloproteinase regulates cancer cell adhesion and locomotion.||Belkin AM,Akimov SS,Zaritskaya LS,Ratnikov BI,Deryugina EI,Strongin AY||The Journal of biological chemistry (276:18415)||2001|
|Not Applicable||Not Cited||Neural bHLH genes control the neuronal versus glial fate decision in cortical progenitors.||Nieto M,Schuurmans C,Britz O,Guillemot F||Neuron (29:401)||2001|
|Not Applicable||Not Cited||Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis.||Botelho RJ,Teruel M,Dierckman R,Anderson R,Wells A,York JD,Meyer T,Grinstein S||The Journal of cell biology (151:1353)||2000|
Interleukin-1beta and tumor necrosis factor-alpha are expressed by different subsets of microglia and macrophages after ischemic stroke in mice.
A-11007 was used in immunohistochemistry to test if IL-1beta and TNF-alpha are synthesized by overlapping or segregated populations of cells after ischemic stroke in mice
|Clausen BH,Lambertsen KL,Babcock AA,Holm TH,Dagnaes-Hansen F,Finsen B||Journal of neuroinflammation (5:null)||2008|
Effects on differentiation of embryonic ventral midbrain progenitors by Lmx1a, Msx1, Ngn2, and Pitx3.
A-11007 was used in immunocytochemistry to elucidate how to induce controlled differentiation of neural stem cells
|Roybon L,Hjalt T,Christophersen NS,Li JY,Brundin P||The Journal of neuroscience : the official journal of the Society for Neuroscience (28:3644)||2008|