Immunofluorescence analysis of Chicken anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 was performed using HeLa cells stained with alpha Tubulin Rabbit Polyclonal Primary Antibody (PA516891). 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 µg/ml Rabbit primary antibody for 3 hours at room temperature. Chicken anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 (A21441) was used at a concentration of 4µg/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 (S36938). F-actin was stained with Rhodamine Phalloidin (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||Rabbit|
|Published species reactivity||Not Applicable|
|Host / Isotype||Chicken / IgY|
|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 human and mouse IgG prior to conjugation|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:500|
|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.
To minimize cross-reactivity, these chicken anti-rabbit IgG (H+L) whole secondary antibodies have been affinity purified and cross-adsorbed against human and mouse IgG prior to conjugation. 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.
Lack of miR-133a Decreases Contractility of Diabetic Hearts: A Role for Novel Cross Talk Between Tyrosine Aminotransferase and Tyrosine Hydroxylase.
A-21441 was used in western blot to test if miR-133a is involved in the crosstalk between tyrosine hydroxylase and tyrosine aminotransferase in the diabetic heart
|Nandi SS,Zheng H,Sharma NM,Shahshahan HR,Patel KP,Mishra PK||Diabetes (65:3075)||2016|
|Not Applicable||Not Cited||TIMMDC1/C3orf1 functions as a membrane-embedded mitochondrial complex I assembly factor through association with the MCIA complex.||Guarani V,Paulo J,Zhai B,Huttlin EL,Gygi SP,Harper JW||Molecular and cellular biology (34:847)||2014|
|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||Filamin B mediates ICAM-1-driven leukocyte transendothelial migration.||Kanters E,van Rijssel J,Hensbergen PJ,Hondius D,Mul FP,Deelder AM,Sonnenberg A,van Buul JD,Hordijk PL||The Journal of biological chemistry (283:31830)||2008|
|Not Applicable||Not Cited||Close relation of arterial ICC-like cells to the contractile phenotype of vascular smooth muscle cell.||Pucovský V,Harhun MI,Povstyan OV,Gordienko DV,Moss RF,Bolton TB||Journal of cellular and molecular medicine (11:764)||2007|
|Not Applicable||Not Cited||Aberrant expression of ID2, a suppressor of B-cell-specific gene expression, in Hodgkin's lymphoma.||Renné C,Martin-Subero JI,Eickernjäger M,Hansmann ML,Küppers R,Siebert R,Bräuninger A||The American journal of pathology (169:655)||2006|
|Not Applicable||Not Cited||EphB receptors regulate dendritic spine morphogenesis through the recruitment/phosphorylation of focal adhesion kinase and RhoA activation.||Moeller ML,Shi Y,Reichardt LF,Ethell IM||The Journal of biological chemistry (281:1587)||2006|
|Not Applicable||Not Cited||Mitochondrial alterations induced by the p13II protein of human T-cell leukemia virus type 1. Critical role of arginine residues.||D'Agostino DM,Ranzato L,Arrigoni G,Cavallari I,Belleudi F,Torrisi MR,Silic-Benussi M,Ferro T,Petronilli V,Marin O,Chieco-Bianchi L,Bernardi P,Ciminale V||The Journal of biological chemistry (277:34424)||2002|
|Not Applicable||Not Cited||
Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics.
A-21441 was used in immunocytochemistry to report the tyrosine phosphorylation events that occur after specific c-Src stimulation
|Ferrando IM,Chaerkady R,Zhong J,Molina H,Jacob HK,Herbst-Robinson K,Dancy BM,Katju V,Bose R,Zhang J,Pandey A,Cole PA||Molecular and cellular proteomics : MCP (11:355)||2012|
|Not Applicable||Not Cited||
Extracellular matrix-induced transforming growth factor-beta receptor signaling dynamics.
A-21441 was used in immunocytochemistry to elucidate TGF-beta receptor dynamics and consequential signaling
|Garamszegi N,Garamszegi SP,Samavarchi-Tehrani P,Walford E,Schneiderbauer MM,Wrana JL,Scully SP||Oncogene (29:2368)||2010|
|Not Applicable||Not Cited||
Mesoscopic hydrogel molding to control the 3D geometry of bioartificial muscle tissues.
A-21441 was used in immunohistochemistry to develop three-dimensional muscle tissue architectures in vitro
|Bian W,Liau B,Badie N,Bursac N||Nature protocols (4:1522)||2009|
|Not Applicable||Not Cited||
Interleukin-1beta and tumor necrosis factor-alpha are expressed by different subsets of microglia and macrophages after ischemic stroke in mice.
A-21441 was used in flow cytometry and immunohistochemistry to test if IL-1beta and TNF-alpha are synthesized by overlapping or segregated populations of cells after ischemic stroke in mice
|Clausen BH,Lambertsen KL,Babcock AA,Holm TH,Dagnaes-Hansen F,Finsen B||Journal of neuroinflammation (5:null)||2008|