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The endogenous alkaline phosphatase enzyme of osteosarcoma cells localized with a mouse anti–rat alkaline phosphatase monoclonal antibody, RBM 211.13, which was visualized with Alexa Fluor® 594 goat anti–mouse IgG, F(ab')<sub>2</sub> fragments (Cat. no. A11020). The blue-fluorescent Hoechst 33342 (Cat. no. H1399, H3570, H21492) nucleic acid stain was used as a counterstain to the red fluorescence of the Alexa Fluor® 594 secondary antibody. The primary antibody was a gift from Dr. Jane Aubin, University of Toronto. The double-exposure image was acquired using longpass filter sets appropriate for the Texas Red® dye and DAPI.
|Tested species reactivity||Mouse|
|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 human IgG and serum|
|Antibody Form||F(ab')2 Fragment|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1-10 µg/mL|
|Immunocytochemistry (ICC)||1-10 µg/mL|
|Immunofluorescence (IF)||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 11 publications below|
To minimize cross-reactivity, these goat anti-mouse IgG (H+L) divalent F(ab')2 secondary antibodies have been affinity purified and cross-adsorbed against human IgG and serum. 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||Spatial distribution of DARPP-32 in dendritic spines.||Blom H,Rönnlund D,Scott L,Westin L,Widengren J,Aperia A,Brismar H||PloS one (8:null)||2013|
|Not Applicable||Not Cited||A reproducible technique for specific labeling of antigens using preformed fluorescent molecular IgG-F(ab')2 complexes from primary antibodies of the same species.||Owen GR,Häkkinen L,Wu C,Larjava H||Microscopy research and technique (73:623)||2010|
|Not Applicable||Not Cited||Polycomb group protein enhancer of zeste 2 is an oncogene that promotes the neoplastic transformation of a benign prostatic epithelial cell line.||Karanikolas BD,Figueiredo ML,Wu L||Molecular cancer research : MCR (7:1456)||2009|
|Not Applicable||Not Cited||Phosphatidylcholine-specific phospholipase C activation in epithelial ovarian cancer cells.||Spadaro F,Ramoni C,Mezzanzanica D,Miotti S,Alberti P,Cecchetti S,Iorio E,Dolo V,Canevari S,Podo F||Cancer research (68:6541)||2008|
|Not Applicable||Not Cited||Molecular identification of a SNAP-25-like SNARE protein in Paramecium.||Schilde C,Lutter K,Kissmehl R,Plattner H||Eukaryotic cell (7:1387)||2008|
|Not Applicable||Not Cited||Regulation of epithelial tight junction assembly and disassembly by AMP-activated protein kinase.||Zheng B,Cantley LC||Proceedings of the National Academy of Sciences of the United States of America (104:819)||2007|
|Not Applicable||Not Cited||Role of H+-ATPase-mediated acidification in sorting and release of the regulated secretory protein chromogranin A: evidence for a vesiculogenic function.||Taupenot L,Harper KL,O'Connor DT||The Journal of biological chemistry (280:3885)||2005|
|Not Applicable||Not Cited||Human eosinophils produce the T cell-attracting chemokines MIG and IP-10 upon stimulation with IFN-gamma.||Dajotoy T,Andersson P,Bjartell A,Löfdahl CG,Tapper H,Egesten A||Journal of leukocyte biology (76:685)||2004|
|Not Applicable||Not Cited||Matrix proteins can generate the higher order architecture of the Golgi apparatus.||Seemann J,Jokitalo E,Pypaert M,Warren G||Nature (407:1022)||2000|
|Not Applicable||Not Cited||Dual targeting property of the N-terminal signal sequence of P4501A1. Targeting of heterologous proteins to endoplasmic reticulum and mitochondria.||Bhagwat SV,Biswas G,Anandatheerthavarada HK,Addya S,Pandak W,Avadhani NG||The Journal of biological chemistry (274:24014)||1999|
|Not Applicable||Not Cited||Redundant and distinct functions for dynamin-1 and dynamin-2 isoforms.||Altschuler Y,Barbas SM,Terlecky LJ,Tang K,Hardy S,Mostov KE,Schmid SL||The Journal of cell biology (143:1871)||1998|