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HeLa cell transfected with pShooter pCMV/myc/mito/GFP, then fixed and permeabilized. Green-fluorescent protein (GFP) localized in the mitochondria was labeled with anti-GFP mouse IgG<sub>2a</sub> (Cat. no. A11120) and detected with orange-fluorescent Alexa Fluor® 555 goat anti–mouse IgG (CAt. no. A21422), which colocalized with the dim GFP fluorescence. F-actin was labeled with green-fluorescent Alexa Fluor® 488 phalloidin (Cat. no. A12379), and the nucleus was stained with blue-fluorescent DAPI (Cat. no. D1306, D3571, D21490). The sample was mounted using ProLong® Gold antifade reagent (Cat. no. P36930). Some GFP fluorescence is retained in the mitochondria after fixation (top), but immunolabeling and detection greatly improve visualization (bottom).
|Tested species reactivity||Mouse|
|Published species reactivity||Not Applicable|
|Host / Isotype||Goat / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 555|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Cross Adsorption||Against human IgG and human serum prior to conjugation|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1-10 µg/mL|
|Immunocytochemistry (ICC)||1-10 µg/ml|
|Immunofluorescence (IF)||1-10 µg/mL|
|Immunohistochemistry (IHC)||1-10 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.
|Miscellaneous PubMed (MISC)||See 22 publications below|
To minimize cross-reactivity, these goat anti-mouse IgG (H+L) whole secondary antibodies have been affinity purified and cross-adsorbed against human IgG 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 555 dye is a bright, orange-fluorescent dye with excitation ideally suited to the 555 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 555 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 555 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||Promoter- and RNA polymerase II-dependent hsp-16 gene association with nuclear pores in Caenorhabditis elegans.||Rohner S,Kalck V,Wang X,Ikegami K,Lieb JD,Gasser SM,Meister P||The Journal of cell biology (200:589)||2013|
|Not Applicable||Not Cited||High-throughput discovery of synthetic surfaces that support proliferation of pluripotent cells.||Derda R,Musah S,Orner BP,Klim JR,Li L,Kiessling LL||Journal of the American Chemical Society (132:1289)||2010|
|Not Applicable||Not Cited||High efficacy of a Listeria-based vaccine against metastatic breast cancer reveals a dual mode of action.||Kim SH,Castro F,Paterson Y,Gravekamp C||Cancer research (69:5860)||2009|
|Not Applicable||Not Cited||Simultaneous detection of mRNA and protein stem cell markers in live cells.||Rhee WJ,Bao G||BMC biotechnology (9:null)||2009|
|Not Applicable||Not Cited||Protective role of endogenous gangliosides for lysosomal pathology in a cellular model of synucleinopathies.||Wei J,Fujita M,Nakai M,Waragai M,Sekigawa A,Sugama S,Takenouchi T,Masliah E,Hashimoto M||The American journal of pathology (174:1891)||2009|
|Not Applicable||Not Cited||Early endosomes and endosomal coatomer are required for autophagy.||Razi M,Chan EY,Tooze SA||The Journal of cell biology (185:305)||2009|
|Not Applicable||Not Cited||Dual roles for an arginine-rich motif in specific genome recognition and localization of viral coat protein to RNA replication sites in flock house virus-infected cells.||Venter PA,Marshall D,Schneemann A||Journal of virology (83:2872)||2009|
|Not Applicable||Not Cited||High-efficiency labeling of sialylated glycoproteins on living cells.||Zeng Y,Ramya TN,Dirksen A,Dawson PE,Paulson JC||Nature methods (6:207)||2009|
|Not Applicable||Not Cited||A fluorimetry-based ssYFP secretion assay to monitor vasopressin-induced exocytosis in LLC-PK1 cells expressing aquaporin-2.||Nunes P,Hasler U,McKee M,Lu HA,Bouley R,Brown D||American journal of physiology. Cell physiology (295:C1476)||2008|
|Not Applicable||Not Cited||The transmembrane domain of the severe acute respiratory syndrome coronavirus ORF7b protein is necessary and sufficient for its retention in the Golgi complex.||Schaecher SR,Diamond MS,Pekosz A||Journal of virology (82:9477)||2008|
|Not Applicable||Not Cited||Rare steroid receptor-negative basal-like tumorigenic cells in luminal subtype human breast cancer xenografts.||Horwitz KB,Dye WW,Harrell JC,Kabos P,Sartorius CA||Proceedings of the National Academy of Sciences of the United States of America (105:5774)||2008|
|Not Applicable||Not Cited||Resolution of de novo HIV production and trafficking in immature dendritic cells.||Turville SG,Aravantinou M,Stössel H,Romani N,Robbiani M||Nature methods (5:75)||2008|
|Not Applicable||Not Cited||In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2.||Arenkiel BR,Peca J,Davison IG,Feliciano C,Deisseroth K,Augustine GJ,Ehlers MD,Feng G||Neuron (54:205)||2007|
|Not Applicable||Not Cited||Mining a yeast library for brain endothelial cell-binding antibodies.||Wang XX,Cho YK,Shusta EV||Nature methods (4:143)||2007|
|Not Applicable||Not Cited||Comparison of hydroxylated print additives on antibody microarray performance.||Wu P,Grainger DW||Journal of proteome research (5:2956)||2006|
|Not Applicable||Not Cited||Phosphatidylinositol-3,4,5-trisphosphate regulates the formation of the basolateral plasma membrane in epithelial cells.||Gassama-Diagne A,Yu W,ter Beest M,Martin-Belmonte F,Kierbel A,Engel J,Mostov K||Nature cell biology (8:963)||2006|
|Not Applicable||Not Cited||Delineation of type I protein kinase A-selective signaling events using an RI anchoring disruptor.||Carlson CR,Lygren B,Berge T,Hoshi N,Wong W,Taskén K,Scott JD||The Journal of biological chemistry (281:21535)||2006|
|Not Applicable||Not Cited||H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks.||Marti TM,Hefner E,Feeney L,Natale V,Cleaver JE||Proceedings of the National Academy of Sciences of the United States of America (103:9891)||2006|
|Not Applicable||Not Cited||Paxillin phosphorylation at Ser273 localizes a GIT1-PIX-PAK complex and regulates adhesion and protrusion dynamics.||Nayal A,Webb DJ,Brown CM,Schaefer EM,Vicente-Manzanares M,Horwitz AR||The Journal of cell biology (173:587)||2006|
|Not Applicable||Not Cited||Microarrays for the functional analysis of the chemical-kinase interactome.||Horiuchi KY,Wang Y,Diamond SL,Ma H||Journal of biomolecular screening (11:48)||2006|
|Not Applicable||Not Cited||Calcineurin localization in skeletal muscle offers insights into potential new targets.||Torgan CE,Daniels MP||The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society (54:119)||2006|
|Not Applicable||Not Cited||Multiplex GPCR assay in reverse transfection cell microarrays.||Mishina YM,Wilson CJ,Bruett L,Smith JJ,Stoop-Myer C,Jong S,Amaral LP,Pedersen R,Lyman SK,Myer VE,Kreider BL,Thompson CM||Journal of biomolecular screening (9:196)||2004|