Immunofluorescence analysis of Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 546 (A10040) 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. Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 546 (A10040) 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||Donkey / IgG|
|Conjugate||Alexa Fluor® 546|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:500|
|Immunocytochemistry (ICC)||4 µg/ml|
|Immunofluorescence (IF)||4 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.
These donkey anti-rabbit IgG (H+L) whole secondary antibodies have been affinity-purified and show minimum cross-reactivity to bovine, chicken, goat, guinea pig, hamster, horse, human, mouse, rat, and sheep serum proteins. 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 may be the presence of endogenous immunoglobulins.
Alexa Fluor dyes are among the most trusted fluorescent dyes available today. Invitrogen™ Alexa Fluor 546 dye is a bright, orange-fluorescent dye with excitation ideally suited to the 546 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 546 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 546 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||
Investigation into the effects of varying frequency of mechanical stimulation in a cycle-by-cycle manner on engineered cardiac construct function.
A10040 was used in immunohistochemistry (frozen) to develop a bioreactor system that allows for the control of the mechanical stimulation of engineered cardiac tissue on a cycle-by-cycle basis.
|Morgan KY,Black LD||Journal of tissue engineering and regenerative medicine (11:342)||2017|
|Not Applicable||Not Cited||Ubiquitylation and activation of a Rab GTPase is promoted by a ß¿AR-HACE1 complex.||Lachance V,Degrandmaison J,Marois S,Robitaille M,Génier S,Nadeau S,Angers S,Parent JL||Journal of cell science (127:111)||2014|
|Not Applicable||Not Cited||Hippo signaling impedes adult heart regeneration.||Heallen T,Morikawa Y,Leach J,Tao G,Willerson JT,Johnson RL,Martin JF||Development (Cambridge, England) (140:4683)||2013|
|Not Applicable||Not Cited||Deficits in adult neurogenesis, contextual fear conditioning, and spatial learning in a Gfap mutant mouse model of Alexander disease.||Hagemann TL,Paylor R,Messing A||The Journal of neuroscience : the official journal of the Society for Neuroscience (33:18698)||2013|
|Not Applicable||Not Cited||Clostridium difficile toxin B-induced necrosis is mediated by the host epithelial cell NADPH oxidase complex.||Farrow MA,Chumbler NM,Lapierre LA,Franklin JL,Rutherford SA,Goldenring JR,Lacy DB||Proceedings of the National Academy of Sciences of the United States of America (110:18674)||2013|
|Not Applicable||Not Cited||Carbachol-induced MUC17 endocytosis is concomitant with NHE3 internalization and CFTR membrane recruitment in enterocytes.||Pelaseyed T,Gustafsson JK,Gustafsson IJ,Ermund A,Hansson GC||American journal of physiology. Cell physiology (305:C457)||2013|
|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||GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes.||Orlandi C,Posokhova E,Masuho I,Ray TA,Hasan N,Gregg RG,Martemyanov KA||The Journal of cell biology (197:711)||2012|
|Not Applicable||Not Cited||Coupling of 6-phosphogluconate dehydrogenase with NADPH oxidase in neutrophils: Nox2 activity regulation by NADPH availability.||Baillet A,Xu R,Grichine A,Berthier S,Morel F,Paclet MH||FASEB journal : official publication of the Federation of American Societies for Experimental Biology (25:2333)||2011|
|Not Applicable||Not Cited||Neuropilin-1 signaling through p130Cas tyrosine phosphorylation is essential for growth factor-dependent migration of glioma and endothelial cells.||Evans IM,Yamaji M,Britton G,Pellet-Many C,Lockie C,Zachary IC,Frankel P||Molecular and cellular biology (31:1174)||2011|
|Not Applicable||Not Cited||Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice.||Pitulescu ME,Schmidt I,Benedito R,Adams RH||Nature protocols (5:1518)||2010|
|Not Applicable||Not Cited||Crucial involvement of the CX3CR1-CX3CL1 axis in dextran sulfate sodium-mediated acute colitis in mice.||Kostadinova FI,Baba T,Ishida Y,Kondo T,Popivanova BK,Mukaida N||Journal of leukocyte biology (88:133)||2010|
|Not Applicable||Not Cited||Identification and validation of novel spinophilin-associated proteins in rodent striatum using an enhanced ex vivo shotgun proteomics approach.||Baucum AJ,Jalan-Sakrikar N,Jiao Y,Gustin RM,Carmody LC,Tabb DL,Ham AJ,Colbran RJ||Molecular and cellular proteomics : MCP (9:1243)||2010|
|Not Applicable||Not Cited||Cisplatin ototoxicity blocks sensory regeneration in the avian inner ear.||Slattery EL,Warchol ME||The Journal of neuroscience : the official journal of the Society for Neuroscience (30:3473)||2010|
|Not Applicable||Not Cited||Urinary-type plasminogen activator receptor/alpha 3 beta 1 integrin signaling, altered gene expression, and oral tumor progression.||Ghosh S,Koblinski J,Johnson J,Liu Y,Ericsson A,Davis JW,Shi Z,Ravosa MJ,Crawford S,Frazier S,Stack MS||Molecular cancer research : MCR (8:145)||2010|
|Not Applicable||Not Cited||An impaired transendothelial migration potential of chronic lymphocytic leukemia (CLL) cells can be linked to ephrin-A4 expression.||Trinidad EM,Ballesteros M,Zuloaga J,Zapata A,Alonso-Colmenar LM||Blood (114:5081)||2009|
|Not Applicable||Not Cited||Sprouty2 interacts with protein kinase C delta and disrupts phosphorylation of protein kinase D1.||Chow SY,Yu CY,Guy GR||The Journal of biological chemistry (284:19623)||2009|
|Not Applicable||Not Cited||Aire-deficient C57BL/6 mice mimicking the common human 13-base pair deletion mutation present with only a mild autoimmune phenotype.||Hubert FX,Kinkel SA,Crewther PE,Cannon PZ,Webster KE,Link M,Uibo R,O'Bryan MK,Meager A,Forehan SP,Smyth GK,Mittaz L,Antonarakis SE,Peterson P,Heath WR,Scott HS||Journal of immunology (Baltimore, Md. : 1950) (182:3902)||2009|
Ventral pallidal projections to mediodorsal thalamus and ventral tegmental area play distinct roles in outcome-specific Pavlovian-instrumental transfer.
A10040 was used in immunohistochemistry - frozen section to study the Pavlovian-instrumental transfer effect of the rostral medial ventral pallidum region innervated by the nucleus accumbens shell
|Leung BK,Balleine BW||The Journal of neuroscience : the official journal of the Society for Neuroscience (35:4953)||2015|