Immunofluorescence analysis of Donkey anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate was performed using A549 cells stained with alpha Tubulin (YL1/2) Rat Monoclonal Antibody (Product # MA1-80017). 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 2µg/ml Rat primary antibody for 3 hours at room temperature. Donkey anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate (Product # A21208) was used at a concentration of 1µg/ml in phosphate buffered saline containing 0.2 % BSA for 45 minutes at room temperature, for detection of alpha Tubulin in the cytoplasm (Panel a: green). Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (Product # S36938). F-actin was stained with Rhodamine Phalloidin (Product # R415, 1:300) (Panel c: red). 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||Rat|
|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|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:500|
|Immunocytochemistry (ICC)||1 µg/ml|
|Immunofluorescence (IF)||1 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-rat 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, rabbit, 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 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.
Genome-wide identification of regulatory elements in Sertoli cells.
A-21208 was used in immunohistochemistry to identify genetic regulatory sites involved in sex determination
|Maatouk DM,Natarajan A,Shibata Y,Song L,Crawford GE,Ohler U,Capel B||Development (Cambridge, England) (144:720)||2017|
Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis.
A-21208 was used in immunohistochemistry to study mouse organogenesis developmental processes using ex vivo upright droplet cultures of whole fetal fetal organs
|Potter SJ,DeFalco T||Journal of visualized experiments : JoVE (null:null)||2015|
Hedgehog signaling promotes basal progenitor expansion and the growth and folding of the neocortex.
A-21208 was used in immunohistochemistry - frozen section to examine Sonic hedgehog signaling in the human fetal neocortex
|Wang L,Hou S,Han YG||Nature neuroscience (19:888)||2016|
Atm deletion with dual recombinase technology preferentially radiosensitizes tumor endothelium.
A-21208 was used in immunohistochemistry - frozen section to test if loss of Atm in endothelial cells sensitizes tumors and normal tissues to radiation.
|Moding EJ,Lee CL,Castle KD,Oh P,Mao L,Zha S,Min HD,Ma Y,Das S,Kirsch DG||The Journal of clinical investigation (124:3325)||2014|
|Not Applicable||10 µg/ml||
Fulminant lymphocytic choriomeningitis virus-induced inflammation of the CNS involves a cytokine-chemokine-cytokine-chemokine cascade.
A-21208 was used in immunohistochemistry - frozen section to address the mechanisms regulating the expression of CXCL10 in the central nervous system of lymphocytic choriomeningitis virus -infected mice
|Christensen JE,Simonsen S,Fenger C,Sørensen MR,Moos T,Christensen JP,Finsen B,Thomsen AR||Journal of immunology (Baltimore, Md. : 1950) (182:1079)||2009|
|Not Applicable||Not Cited||
A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation.
A-21208 was used in immunocytochemistry to characterize mammalian nuclear RNA N6-adenosine methylation mediation by a METTL3-METTL14 complex
|Liu J,Yue Y,Han D,Wang X,Fu Y,Zhang L,Jia G,Yu M,Lu Z,Deng X,Dai Q,Chen W,He C||Nature chemical biology (10:93)||2014|
|Not Applicable||Not Cited||
Dissociation of cytokinesis initiation from mitotic control in a eukaryote.
A-21208 was used in immunocytochemistry to investigate cytokinesis initiation in the trypanosome
|Kumar P,Wang CC||Eukaryotic cell (5:92)||2006|
N6-methyladenosine-dependent regulation of messenger RNA stability.
A-21208 was used in immunocytochemistry to elucidate the function of m(6)A modifications.
|Wang X,Lu Z,Gomez A,Hon GC,Yue Y,Han D,Fu Y,Parisien M,Dai Q,Jia G,Ren B,Pan T,He C||Nature (505:117)||2014|
|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||Deficient ghrelin receptor-mediated signaling compromises thymic stromal cell microenvironment by accelerating thymic adiposity.||Youm YH,Yang H,Sun Y,Smith RG,Manley NR,Vandanmagsar B,Dixit VD||The Journal of biological chemistry (284:7068)||2009|
|Not Applicable||Not Cited||Medullary thymic epithelial cells expressing Aire represent a unique lineage derived from cells expressing claudin.||Hamazaki Y,Fujita H,Kobayashi T,Choi Y,Scott HS,Matsumoto M,Minato N||Nature immunology (8:304)||2007|
|Not Applicable||Not Cited||Involvement of SUMO modification in MBD1- and MCAF1-mediated heterochromatin formation.||Uchimura Y,Ichimura T,Uwada J,Tachibana T,Sugahara S,Nakao M,Saitoh H||The Journal of biological chemistry (281:23180)||2006|
|Not Applicable||Not Cited||Novel role for RbAp48 in tissue-specific, estrogen deficiency-dependent apoptosis in the exocrine glands.||Ishimaru N,Arakaki R,Omotehara F,Yamada K,Mishima K,Saito I,Hayashi Y||Molecular and cellular biology (26:2924)||2006|
|Not Applicable||Not Cited||LPS receptor (CD14): a receptor for phagocytosis of Alzheimer's amyloid peptide.||Liu Y,Walter S,Stagi M,Cherny D,Letiembre M,Schulz-Schaeffer W,Heine H,Penke B,Neumann H,Fassbender K||Brain : a journal of neurology (128:1778)||2005|
|Not Applicable||Not Cited||Intracellular Helicobacter pylori in gastric epithelial progenitors.||Oh JD,Karam SM,Gordon JI||Proceedings of the National Academy of Sciences of the United States of America (102:5186)||2005|