Flow cytometry analysis of Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647 (A31573) was performed using K-562 cells stained with FOXN3 ABfinity™ Rabbit Monoclonal Antibody (702555). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled withFOXN3 ABfinity™ Rabbit Monoclonal Antibody or with rabbit isotype control at 3-5 ug/million cells in 2.5% BSA and incubated for 2 hours at room temperature. The cells were then labeled with Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647 (A31573) at a dilution of 1:500 for 1 hour at room temperature. A representative 10,000 cells were acquired and analyzed for each sample using the Attune® NxT Acoustic Focusing Cytometer. Panels a, b and c represent cells stained with the secondary antibody alone, isotype control andFOXN3 ABfinity™ Rabbit Monoclonal Antibody respectively. Median fluorescence intensity from the three samples is compared in panel d.
|Tested species reactivity||Rabbit|
|Published species reactivity||Not Applicable|
|Host / Isotype||Donkey / 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|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:500|
|Immunocytochemistry (ICC)||1-10 µg/ml|
|Immunofluorescence (IF)||1-10 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.
To minimize cross-reactivity, these donkey anti-rabbit IgG whole antibodies have been affinity-purified and show a published cross-reactivity to rat IgG. 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 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.
Murine neural crest stem cells and embryonic stem cell-derived neuron precursors survive and differentiate after transplantation in a model of dorsal root avulsion.
A-31573 was used in immunohistochemistry - paraffin section to compare the survival and migration of murine boundary cap neural crest stem cells and predifferentiated neuron precursors after their implantation
|Konig N,Trolle C,Kapuralin K,Adameyko I,Mitrecic D,Aldskogius H,Shortland PJ,Kozlova EN||Journal of tissue engineering and regenerative medicine (11:129)||2017|
|Not Applicable||Not Cited||
Effect of Polymer Hydration State on In-Gel Immunoassays.
A-31573 was used in western blot to study the effect of polymer hydration state in regards to in-gel immunoassays
|Vlassakis J,Herr AE||Analytical chemistry (87:11030)||2015|
Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis.
A-31573 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|
Congenital heart disease protein 5 associates with CASZ1 to maintain myocardial tissue integrity.
A-31573 was used in immunohistochemistry to investigate the role of cardiac transcription factor CASTOR (CASZ1) in heart development
|Sojka S,Amin NM,Gibbs D,Christine KS,Charpentier MS,Conlon FL||Development (Cambridge, England) (141:3040)||2014|
|Not Applicable||Not Cited||EdU, a new thymidine analogue for labelling proliferating cells in the nervous system.||Chehrehasa F,Meedeniya AC,Dwyer P,Abrahamsen G,Mackay-Sim A||Journal of neuroscience methods (177:122)||2009|
|Not Applicable||Not Cited||pLG72 modulates intracellular D-serine levels through its interaction with D-amino acid oxidase: effect on schizophrenia susceptibility.||Sacchi S,Bernasconi M,Martineau M,Mothet JP,Ruzzene M,Pilone MS,Pollegioni L,Molla G||The Journal of biological chemistry (283:22244)||2008|
|Not Applicable||Not Cited||A stochastic mechanism for biofilm formation by Mycoplasma pulmonis.||Simmons WL,Bolland JR,Daubenspeck JM,Dybvig K||Journal of bacteriology (189:1905)||2007|
|Not Applicable||Not Cited||Evidence for stroke-induced neurogenesis in the human brain.||Jin K,Wang X,Xie L,Mao XO,Zhu W,Wang Y,Shen J,Mao Y,Banwait S,Greenberg DA||Proceedings of the National Academy of Sciences of the United States of America (103:13198)||2006|
|Not Applicable||Not Cited||Rotavirus NSP4 induces a novel vesicular compartment regulated by calcium and associated with viroplasms.||Berkova Z,Crawford SE,Trugnan G,Yoshimori T,Morris AP,Estes MK||Journal of virology (80:6061)||2006|
|Not Applicable||Not Cited||TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells.||James D,Levine AJ,Besser D,Hemmati-Brivanlou A||Development (Cambridge, England) (132:1273)||2005|