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Four-color fluorescence in situ hybridization on a Drosophila embryo. A late blastoderm stage (nuclear cycle 14) embryo was probed with four different RNA probes. Blue: sog labeled with DNP, followed by a rabbit anti-dinitrophenyl-KLH IgG antibody (Cat. No. A6430) detected with an Alexa Fluor® 647 chicken anti-rabbit IgG antibody (Cat. No. A21443). Green: ind labeled with biotin, followed by streptavidin HRP and Alexa Fluor® 350 tyramide (TSA Kit #27, Cat. No. T20937). Red: msh labeled with digoxigenin followed by sheep anti-digoxigenin antibody detected with an Alexa Fluor® 488 donkey anti-sheep IgG antibody (Cat. No. A11015). Yellow: sna labeled with fluorescein followed by mouse anti-fluorescein antibody detected with an Alexa Fluor® 555 goat anti-mouse IgG antibody (Cat. No. A21424). Image contributed by Dave Kosman and Ethan Bier, University of California, San Diego.
|Tested species reactivity||Sheep|
|Published species reactivity||Not Applicable|
|Host / Isotype||Donkey / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 488|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Cross Adsorption||Against mouse, rabbit, bovine and human sera and human IgG|
|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|
* 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 donkey anti-sheep IgG (H+L) whole secondary antibodies have been affinity purified and cross-adsorbed against mouse, rabbit, bovine, and human sera, and human IgG. 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 488 dye is a bright, green-fluorescent dye with excitation ideally suited to the 488 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 488 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 488 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||LY2228820 dimesylate, a selective inhibitor of p38 mitogen-activated protein kinase, reduces angiogenic endothelial cord formation in vitro and in vivo.||Tate CM,Blosser W,Wyss L,Evans G,Xue Q,Pan Y,Stancato L||The Journal of biological chemistry (288:6743)||2013|
|Not Applicable||Not Cited||Somatic mutations in CCK2R alter receptor activity that promote oncogenic phenotypes.||Willard MD,Lajiness ME,Wulur IH,Feng B,Swearingen ML,Uhlik MT,Kinzler KW,Velculescu VE,Sjöblom T,Markowitz SD,Powell SM,Vogelstein B,Barber TD||Molecular cancer research : MCR (10:739)||2012|
|Not Applicable||Not Cited||Reducing the multidimensionality of high-content screening into versatile powerful descriptors.||Gorenstein J,Zack B,Marszalek JR,Bagchi A,Subramaniam S,Carroll P,Elbi C||BioTechniques (49:663)||2010|
|Not Applicable||Not Cited||Differential modulation of Akt/glycogen synthase kinase-3beta pathway regulates apoptotic and cytoprotective signaling responses.||Nair VD,Olanow CW||The Journal of biological chemistry (283:15469)||2008|
|Not Applicable||Not Cited||Stimulation of endogenous neurogenesis by anti-EFRH immunization in a transgenic mouse model of Alzheimer's disease.||Becker M,Lavie V,Solomon B||Proceedings of the National Academy of Sciences of the United States of America (104:1691)||2007|
|Not Applicable||Not Cited||p53 mediates nontranscriptional cell death in dopaminergic cells in response to proteasome inhibition.||Nair VD,McNaught KS,González-Maeso J,Sealfon SC,Olanow CW||The Journal of biological chemistry (281:39550)||2006|
|Not Applicable||Not Cited||DNA replication origin plasticity and perturbed fork progression in human inverted repeats.||Lebofsky R,Bensimon A||Molecular and cellular biology (25:6789)||2005|
|Not Applicable||Not Cited||Androgen receptor signaling: mechanism of interleukin-6 inhibition.||Jia L,Choong CS,Ricciardelli C,Kim J,Tilley WD,Coetzee GA||Cancer research (64:2619)||2004|
|Not Applicable||Not Cited||Neurabins recruit protein phosphatase-1 and inhibitor-2 to the actin cytoskeleton.||Terry-Lorenzo RT,Elliot E,Weiser DC,Prickett TD,Brautigan DL,Shenolikar S||The Journal of biological chemistry (277:46535)||2002|
|Not Applicable||Not Cited||Two distinct regions in a yeast myosin-V tail domain are required for the movement of different cargoes.||Catlett NL,Duex JE,Tang F,Weisman LS||The Journal of cell biology (150:513)||2000|
|Not Applicable||Not Cited||AP-4, a novel protein complex related to clathrin adaptors.||Dell'Angelica EC,Mullins C,Bonifacino JS||The Journal of biological chemistry (274:7278)||1999|