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Flow cytometry analysis of Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 680 (A21076) 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 with FOXN3 ABfinity™ 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 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 680 (A21076) 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 and alpha Tubulin 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||Goat / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 680|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Cross Adsorption||Against human IgG, human serum, mouse IgG, mouse serum and bovine serum|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:500|
|Immunofluorescence (IF)||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 19 publications below|
To minimize cross-reactivity, these goat anti-rabbit IgG (H+L) whole secondary antibodies have been affinity purified and cross-adsorbed against human IgG, human serum, mouse IgG, mouse serum, and bovine 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 680 dye is a bright, near-infrared-fluorescent dye with excitation ideally suited to the 680 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 680 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 680 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||Characterization of human-induced pluripotent stem cell-derived cardiomyocytes: bioenergetics and utilization in safety screening.||Rana P,Anson B,Engle S,Will Y||Toxicological sciences : an official journal of the Society of Toxicology (130:117)||2012|
|Not Applicable||Not Cited||Potent and selective inhibition of polycythemia by the quinoxaline JAK2 inhibitor NVP-BSK805.||Baffert F,Régnier CH,De Pover A,Pissot-Soldermann C,Tavares GA,Blasco F,Brueggen J,Chène P,Drueckes P,Erdmann D,Furet P,Gerspacher M,Lang M,Ledieu D,Nolan L,Ruetz S,Trappe J,Vangrevelinghe E,Wartmann M,Wyder L,Hofmann F,Radimerski T||Molecular cancer therapeutics (9:1945)||2010|
|Not Applicable||Not Cited||Nucleotide excision repair-induced H2A ubiquitination is dependent on MDC1 and RNF8 and reveals a universal DNA damage response.||Marteijn JA,Bekker-Jensen S,Mailand N,Lans H,Schwertman P,Gourdin AM,Dantuma NP,Lukas J,Vermeulen W||The Journal of cell biology (186:835)||2009|
|Not Applicable||Not Cited||Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins.||McNaughton BR,Cronican JJ,Thompson DB,Liu DR||Proceedings of the National Academy of Sciences of the United States of America (106:6111)||2009|
|Not Applicable||Not Cited||Glucokinase thermolability and hepatic regulatory protein binding are essential factors for predicting the blood glucose phenotype of missense mutations.||Pino MF,Kim KA,Shelton KD,Lindner J,Odili S,Li C,Collins HW,Shiota M,Matschinsky FM,Magnuson MA||The Journal of biological chemistry (282:13906)||2007|
|Not Applicable||Not Cited||Tumor necrosis factor receptor 2 signaling induces selective c-IAP1-dependent ASK1 ubiquitination and terminates mitogen-activated protein kinase signaling.||Zhao Y,Conze DB,Hanover JA,Ashwell JD||The Journal of biological chemistry (282:7777)||2007|
|Not Applicable||Not Cited||Cell-surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2.||Zehe C,Engling A,Wegehingel S,Schäfer T,Nickel W||Proceedings of the National Academy of Sciences of the United States of America (103:15479)||2006|
|Not Applicable||Not Cited||Serotonin-, protein kinase C-, and Hic-5-associated redistribution of the platelet serotonin transporter.||Carneiro AM,Blakely RD||The Journal of biological chemistry (281:24769)||2006|
|Not Applicable||Not Cited||Protein phosphatase 6 subunit with conserved Sit4-associated protein domain targets IkappaBepsilon.||Stefansson B,Brautigan DL||The Journal of biological chemistry (281:22624)||2006|
|Not Applicable||Not Cited||Binding to Rab3A-interacting molecule RIM regulates the presynaptic recruitment of Munc13-1 and ubMunc13-2.||Andrews-Zwilling YS,Kawabe H,Reim K,Varoqueaux F,Brose N||The Journal of biological chemistry (281:19720)||2006|
|Not Applicable||Not Cited||Negative regulation of Fc epsilonRI-mediated signaling and mast cell function by the adaptor protein LAX.||Zhu M,Rhee I,Liu Y,Zhang W||The Journal of biological chemistry (281:18408)||2006|
|Not Applicable||Not Cited||Congenital progressive hydronephrosis (cph) is caused by an S256L mutation in aquaporin-2 that affects its phosphorylation and apical membrane accumulation.||McDill BW,Li SZ,Kovach PA,Ding L,Chen F||Proceedings of the National Academy of Sciences of the United States of America (103:6952)||2006|
|Not Applicable||Not Cited||Pharmacological promotion of inclusion formation: a therapeutic approach for Huntington's and Parkinson's diseases.||Bodner RA,Outeiro TF,Altmann S,Maxwell MM,Cho SH,Hyman BT,McLean PJ,Young AB,Housman DE,Kazantsev AG||Proceedings of the National Academy of Sciences of the United States of America (103:4246)||2006|
|Not Applicable||Not Cited||Conserved oligomeric Golgi complex subunit 1 deficiency reveals a previously uncharacterized congenital disorder of glycosylation type II.||Foulquier F,Vasile E,Schollen E,Callewaert N,Raemaekers T,Quelhas D,Jaeken J,Mills P,Winchester B,Krieger M,Annaert W,Matthijs G||Proceedings of the National Academy of Sciences of the United States of America (103:3764)||2006|
|Not Applicable||Not Cited||The glycosphingolipid globotriaosylceramide in the metastatic transformation of colon cancer.||Kovbasnjuk O,Mourtazina R,Baibakov B,Wang T,Elowsky C,Choti MA,Kane A,Donowitz M||Proceedings of the National Academy of Sciences of the United States of America (102:19087)||2005|
|Not Applicable||Not Cited||Connexin 43 interacts with zona occludens-1 and -2 proteins in a cell cycle stage-specific manner.||Singh D,Solan JL,Taffet SM,Javier R,Lampe PD||The Journal of biological chemistry (280:30416)||2005|
|Not Applicable||Not Cited||Functional overexpression of gamma-secretase reveals protease-independent trafficking functions and a critical role of lipids for protease activity.||Wrigley JD,Schurov I,Nunn EJ,Martin AC,Clarke EE,Ellis S,Bonnert TP,Shearman MS,Beher D||The Journal of biological chemistry (280:12523)||2005|
|Not Applicable||Not Cited||Elimination of protein kinase MK5/PRAK activity by targeted homologous recombination.||Shi Y,Kotlyarov A,Laabeta K,Gruber AD,Butt E,Marcus K,Meyer HE,Friedrich A,Volk HD,Gaestel M||Molecular and cellular biology (23:7732)||2003|
|Not Applicable||Not Cited||EphB/syndecan-2 signaling in dendritic spine morphogenesis.||Ethell IM,Irie F,Kalo MS,Couchman JR,Pasquale EB,Yamaguchi Y||Neuron (31:1001)||2001|