Immunofluorescence analysis of Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor® 555 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. Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor® 555 (Product # A21434) was used at a concentration of 2µg/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: red). Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (Product # S36938). F-actin was stained with Alexa Fluor® 488 Phalloidin (Product # 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||Rat|
|Published species reactivity||Not Applicable|
|Host / Isotype||Goat / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 555|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Cross Adsorption||Against mouse IgG, mouse serum and human serum prior to conjugation|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:500|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 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.
|Miscellaneous PubMed (MISC)||See 11 publications below|
To minimize cross-reactivity, these goat anti-rat IgG (H+L) whole secondary antibodies have been affinity purified and cross-adsorbed against mouse IgG, mouse serum, and human serum 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 555 dye is a bright, orange-fluorescent dye with excitation ideally suited to the 555 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 555 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 555 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||Dual effects of resveratrol on arterial damage induced by insulin resistance in aged mice.||Baron S,Bedarida T,Cottart CH,Vibert F,Vessieres E,Ayer A,Henrion D,Hommeril B,Paul JL,Renault G,Saubamea B,Beaudeux JL,Procaccio V,Nivet-Antoine V||The journals of gerontology. Series A, Biological sciences and medical sciences (69:260)||2014|
|Not Applicable||Not Cited||Identifying the initiating events of anti-Listeria responses using mice with conditional loss of IFN-¿ receptor subunit 1 (IFNGR1).||Lee SH,Carrero JA,Uppaluri R,White JM,Archambault JM,Lai KS,Chan SR,Sheehan KC,Unanue ER,Schreiber RD||Journal of immunology (Baltimore, Md. : 1950) (191:4223)||2013|
|Not Applicable||Not Cited||ß1 integrin regulates Arg to promote invadopodial maturation and matrix degradation.||Beaty BT,Sharma VP,Bravo-Cordero JJ,Simpson MA,Eddy RJ,Koleske AJ,Condeelis J||Molecular biology of the cell (24:1661)||2013|
|Not Applicable||Not Cited||C5a receptor signaling prevents folate deficiency-induced neural tube defects in mice.||Denny KJ,Coulthard LG,Jeanes A,Lisgo S,Simmons DG,Callaway LK,Wlodarczyk B,Finnell RH,Woodruff TM,Taylor SM||Journal of immunology (Baltimore, Md. : 1950) (190:3493)||2013|
|Not Applicable||Not Cited||Protective anti-inflammatory effect of ADAMTS13 on myocardial ischemia/reperfusion injury in mice.||De Meyer SF,Savchenko AS,Haas MS,Schatzberg D,Carroll MC,Schiviz A,Dietrich B,Rottensteiner H,Scheiflinger F,Wagner DD||Blood (120:5217)||2012|
|Not Applicable||Not Cited||Protective effects of dispersive viscoelastics on corneal endothelial damage in a toxic anterior segment syndrome animal model.||Song JS,Heo JH,Kim HM||Investigative ophthalmology and visual science (53:6164)||2012|
|Not Applicable||Not Cited||Respiratory syncytial virus potentiates ABCA3 mutation-induced loss of lung epithelial cell differentiation.||Kaltenborn E,Kern S,Frixel S,Fragnet L,Conzelmann KK,Zarbock R,Griese M||Human molecular genetics (21:2793)||2012|
|Not Applicable||Not Cited||N-methyl-D-aspartate (NMDA) receptor composition modulates dendritic spine morphology in striatal medium spiny neurons.||Vastagh C,Gardoni F,Bagetta V,Stanic J,Zianni E,Giampà C,Picconi B,Calabresi P,Di Luca M||The Journal of biological chemistry (287:18103)||2012|
|Not Applicable||Not Cited||Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme.||Sarkar AA,Zohn IE||The Journal of cell biology (196:789)||2012|
|Not Applicable||Not Cited||Antibody to Langerin/CD207 localizes large numbers of CD8alpha+ dendritic cells to the marginal zone of mouse spleen.||Idoyaga J,Suda N,Suda K,Park CG,Steinman RM||Proceedings of the National Academy of Sciences of the United States of America (106:1524)||2009|
|Not Applicable||Not Cited||A drosophila model for amyotrophic lateral sclerosis reveals motor neuron damage by human SOD1.||Watson MR,Lagow RD,Xu K,Zhang B,Bonini NM||The Journal of biological chemistry (283:24972)||2008|