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Immunofluorescence analysis of Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 633 (A21071) was performed using HepG2 cells stained with alpha-1 antitrypsin Rabbit Polyclonal Primary Antibody (PA516661). 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 2 ug/ml of rabbit primary antibody for 3 hours at room temperature. Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 633 (A21071) was used at a concentration of 4ug/ml in phosphate buffered saline containing 0.2 % BSA for 45 minutes at room temperature, for detection of alpha-1 antitrypsin in the cytoplasm (Panel a: red). Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (S36938). F-actin was stained with Alexa Fluor® 488 Phalloidin (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||Rabbit|
|Published species reactivity||Not Applicable|
|Host / Isotype||Goat / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 633|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Cross Adsorption||Against bovine IgG, goat IgG, mouse IgG, rat IgG and human IgG|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1-10 µg/mL|
|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-rabbit IgG (H+L) whole secondary antibodies have been affinity purified and cross-adsorbed against bovine IgG, goat IgG, mouse IgG, rat IgG, 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 633 dye is a bright, far-red-fluorescent dye with excitation ideally suited to the 633 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 633 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 633 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.
Assembly Dynamics and Stoichiometry of the Apoptosis Signal-regulating Kinase (ASK) Signalosome in Response to Electrophile Stress.
A-21071 was used in immunocytochemistry to characterize the response to electrophile stress by assembly stoichiometry and dynamics of the apoptosis signal-regulating kinase (ASK) signalosome
|Federspiel JD,Codreanu SG,Palubinsky AM,Winland AJ,Betanzos CM,McLaughlin B,Liebler DC||Molecular and cellular proteomics : MCP (15:1947)||2016|
Foxg1 has an essential role in postnatal development of the dentate gyrus.
A-21071 was used in immunohistochemistry - frozen section to determine the function of Foxg1 in postnatal dentate gyrus neurogenesis.
|Tian C,Gong Y,Yang Y,Shen W,Wang K,Liu J,Xu B,Zhao J,Zhao C||The Journal of neuroscience : the official journal of the Society for Neuroscience (32:2931)||2012|
|Not Applicable||Not Cited||Modulation of cell surface protein free thiols: a potential novel mechanism of action of the sesquiterpene lactone parthenolide.||Skalska J,Brookes PS,Nadtochiy SM,Hilchey SP,Jordan CT,Guzman ML,Maggirwar SB,Briehl MM,Bernstein SH||PloS one (4:null)||2009|
|Not Applicable||Not Cited||Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.||Narendra D,Tanaka A,Suen DF,Youle RJ||The Journal of cell biology (183:795)||2008|
|Not Applicable||Not Cited||Counting contacts between neurons in 3D in confocal laser scanning images.||Wouterlood FG,Boekel AJ,Kajiwara R,Beliën JA||Journal of neuroscience methods (171:296)||2008|
|Not Applicable||Not Cited||Stimulus-dependent regulation of the phagocyte NADPH oxidase by a VAV1, Rac1, and PAK1 signaling axis.||Roepstorff K,Rasmussen I,Sawada M,Cudre-Maroux C,Salmon P,Bokoch G,van Deurs B,Vilhardt F||The Journal of biological chemistry (283:7983)||2008|
|Not Applicable||Not Cited||Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2.||Chubykin AA,Atasoy D,Etherton MR,Brose N,Kavalali ET,Gibson JR,Südhof TC||Neuron (54:919)||2007|
|Not Applicable||Not Cited||Generation of functional hemangioblasts from human embryonic stem cells.||Lu SJ,Feng Q,Caballero S,Chen Y,Moore MA,Grant MB,Lanza R||Nature methods (4:501)||2007|
|Not Applicable||Not Cited||One-step analysis of protein complexes in microliters of cell lysate using indirect immunolabeling and fluorescence cross-correlation spectroscopy.||Stoevesandt O,Brock R||Nature protocols (1:223)||2007|
|Not Applicable||Not Cited||Internalization of novel non-viral vector TAT-streptavidin into human cells.||Rinne J,Albarran B,Jylhävä J,Ihalainen TO,Kankaanpää P,Hytönen VP,Stayton PS,Kulomaa MS,Vihinen-Ranta M||BMC biotechnology (7:null)||2007|
|Not Applicable||Not Cited||Intercellular spreading of Porphyromonas gingivalis infection in primary gingival epithelial cells.||Yilmaz O,Verbeke P,Lamont RJ,Ojcius DM||Infection and immunity (74:703)||2006|
|Not Applicable||Not Cited||Stem cell division is regulated by the microRNA pathway.||Hatfield SD,Shcherbata HR,Fischer KA,Nakahara K,Carthew RW,Ruohola-Baker H||Nature (435:974)||2005|
|Not Applicable||Not Cited||Nuclear transport of single molecules: dwell times at the nuclear pore complex.||Kubitscheck U,Grünwald D,Hoekstra A,Rohleder D,Kues T,Siebrasse JP,Peters R||The Journal of cell biology (168:233)||2005|
|Not Applicable||Not Cited||Vav1 and Ly-GDI two regulators of Rho GTPases, function cooperatively as signal transducers in T cell antigen receptor-induced pathways.||Groysman M,Hornstein I,Alcover A,Katzav S||The Journal of biological chemistry (277:50121)||2002|
|Not Applicable||Not Cited||ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts.||Tsuji T,Ishizaki T,Okamoto M,Higashida C,Kimura K,Furuyashiki T,Arakawa Y,Birge RB,Nakamoto T,Hirai H,Narumiya S||The Journal of cell biology (157:819)||2002|
|Not Applicable||Not Cited||The scaffold protein gravin (cAMP-dependent protein kinase-anchoring protein 250) binds the beta 2-adrenergic receptor via the receptor cytoplasmic Arg-329 to Leu-413 domain and provides a mobile scaffold during desensitization.||Fan G,Shumay E,Wang H,Malbon CC||The Journal of biological chemistry (276:24005)||2001|