U2OS cells were transduced using an adenoviral construct expressing mCherry.<br /> A) Native expression of mCherry detected post-transduction using Texas Red filters (562 nm/624 nm)<br /> B) Anti-Cherry antibody added and cells imaged using the Cy5 filter set (628 nm/692 nm)<br /> C) mCherry expression detected by adding anti-mCherry and Alexa Fluor® 647 goat anti-rat (A21247)
|Tested species reactivity||Rat|
|Published species reactivity||Not Applicable|
|Host / Isotype||Goat / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 647|
|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 *|
|Immunocytochemistry (ICC)||1-10 µg/ml|
|Immunofluorescence (IF)||1-10 µg/mL|
|Immunohistochemistry (IHC)||1-10 µg/ml|
|Western Blot (WB)||Assay Dependent|
* 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-rat IgG (H+L) whole secondary antibodies have been affinity purified and cross-adsorbed against mouse IgG, mouse serum, and human serum. 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 647 dye is a near-infrared-fluorescent dye with excitation ideally suited to the 647 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 647 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 647 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.
Applied stretch initiates directional invasion through the action of Rap1 GTPase as a tension sensor.
A-21247 was used in immunocytochemistry to elucidate initiation of directional invasion via the action of Rap1 GTPase as a tension sensor by applied stretch
|Freeman SA,Christian S,Austin P,Iu I,Graves ML,Huang L,Tang S,Coombs D,Gold MR,Roskelley CD||Journal of cell science (130:152)||2017|
|Not Applicable||Not Cited||
Regulation of transgenes in three-dimensional cultures of primary mouse mammary cells demonstrates oncogene dependence and identifies cells that survive deinduction.
A-21247 was used in immunocytochemistry to develop a three-dimensional culture system and examine the responses of primary mouse mammary epithelial cells to the induction and deinduction of oncogenes
|Jechlinger M,Podsypanina K,Varmus H||Genes and development (23:1677)||2009|
Atm deletion with dual recombinase technology preferentially radiosensitizes tumor endothelium.
A-21247 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||Not Cited||The NALP3 inflammasome is involved in the innate immune response to amyloid-beta.||Halle A,Hornung V,Petzold GC,Stewart CR,Monks BG,Reinheckel T,Fitzgerald KA,Latz E,Moore KJ,Golenbock DT||Nature immunology (9:857)||2008|
|Not Applicable||Not Cited||Direct priming of antiviral CD8+ T cells in the peripheral interfollicular region of lymph nodes.||Hickman HD,Takeda K,Skon CN,Murray FR,Hensley SE,Loomis J,Barber GN,Bennink JR,Yewdell JW||Nature immunology (9:155)||2008|
|Not Applicable||Not Cited||Glutathione export during apoptosis requires functional multidrug resistance-associated proteins.||Hammond CL,Marchan R,Krance SM,Ballatori N||The Journal of biological chemistry (282:14337)||2007|
|Not Applicable||Not Cited||Effect of mannose chain length on targeting of glucocerebrosidase for enzyme replacement therapy of Gaucher disease.||Van Patten SM,Hughes H,Huff MR,Piepenhagen PA,Waire J,Qiu H,Ganesa C,Reczek D,Ward PV,Kutzko JP,Edmunds T||Glycobiology (17:467)||2007|
|Not Applicable||Not Cited||Asymmetric T lymphocyte division in the initiation of adaptive immune responses.||Chang JT,Palanivel VR,Kinjyo I,Schambach F,Intlekofer AM,Banerjee A,Longworth SA,Vinup KE,Mrass P,Oliaro J,Killeen N,Orange JS,Russell SM,Weninger W,Reiner SL||Science (New York, N.Y.) (315:1687)||2007|
|Not Applicable||Not Cited||The contribution of bone marrow-derived cells to the development of renal interstitial fibrosis.||Li J,Deane JA,Campanale NV,Bertram JF,Ricardo SD||Stem cells (Dayton, Ohio) (25:697)||2007|
|Not Applicable||Not Cited||The strategy of T cell antigen-presenting cell encounter in antigen-draining lymph nodes revealed by imaging of initial T cell activation.||Bajénoff M,Granjeaud S,Guerder S||The Journal of experimental medicine (198:715)||2003|