Flow cytometry analysis of Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 (Product # A11077) was performed using K-562 cells stained with alpha Tubulin Rat Monoclonal Antibody (Product # MA1-80017). 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 alpha Tubulin antibody (red histogram) or with rat isotype control (pink histogram) at 3-5 µg/million cells in 2.5% BSA and incubated for 2 hours at room temperature. The cells were then labeled with Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 (Product # A11077) 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. The green histogram represents no-primary-antibody control.
|Tested species reactivity||Rat|
|Published species reactivity||Not Applicable|
|Host / Isotype||Goat / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 568|
|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)||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 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 568 dye is a bright, orange/red-fluorescent dye with excitation ideally suited to the 568 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 568 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 568 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.
High-performance probes for light and electron microscopy.
A-11077 was used in immunohistochemistry to develop and characterize 'spaghetti monster' fluorescent proteins
|Viswanathan S,Williams ME,Bloss EB,Stasevich TJ,Speer CM,Nern A,Pfeiffer BD,Hooks BM,Li WP,English BP,Tian T,Henry GL,Macklin JJ,Patel R,Gerfen CR,Zhuang X,Wang Y,Rubin GM,Looger LL||Nature methods (12:568)||2015|
Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila.
A-11077 was used in immunohistochemistry to study the Atg17/FIP200 complex that localizes to perilysosomal Ref(2)P aggregates and activates Atg1 in Drosophila that promotes autophagy
|Nagy P,Kárpáti M,Varga A,Pircs K,Venkei Z,Takáts S,Varga K,Erdi B,Heged¿s K,Juhász G||Autophagy (10:453)||2014|
Expansion of the gateway multisite recombination cloning toolkit.
A-11077 was used in immunohistochemistry to expand the Gateway MultiSite cloning system
|Shearin HK,Dvarishkis AR,Kozeluh CD,Stowers RS||PloS one (8:null)||2013|
A Gateway MultiSite recombination cloning toolkit.
A-11077 was used in immunohistochemistry to develop a model system-independent toolkit for multisite recombination using the Gateway system
|Petersen LK,Stowers RS||PloS one (6:null)||2011|
Interleukin-1beta and tumor necrosis factor-alpha are expressed by different subsets of microglia and macrophages after ischemic stroke in mice.
A-11077 was used in 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|
An epigenetic feedback regulatory loop involving microRNA-195 and MBD1 governs neural stem cell differentiation.
A-11077 was used in immunocytochemistry to report that MBD1 deficiency in adult neural stem/progenitor cells results in altered expression of several noncoding microRNAs
|Liu C,Teng ZQ,McQuate AL,Jobe EM,Christ CC,von Hoyningen-Huene SJ,Reyes MD,Polich ED,Xing Y,Li Y,Guo W,Zhao X||PloS one (8:null)||2013|
Cross talk between microRNA and epigenetic regulation in adult neurogenesis.
A-11077 was used in immunocytochemistry to investigate the cross talk between miR-137 and epigenetic regulation in adult neural stem cells.
|Szulwach KE,Li X,Smrt RD,Li Y,Luo Y,Lin L,Santistevan NJ,Li W,Zhao X,Jin P||The Journal of cell biology (189:127)||2010|
|Not Applicable||Not Cited||
NK receptors, Substance P, Ano1 expression and ultrastructural features of the muscle coat in Cav-1(-/-) mouse ileum.
A-11077 was used in immunohistochemistry - frozen section to use caveolin-1 knockout mice to investigate smooth muscle cells, interstitial cells of Cajal, and neuronal morphology in the intestine.
|Cipriani G,Serboiu CS,Gherghiceanu M,Faussone-Pellegrini MS,Vannucchi MG||Journal of cellular and molecular medicine (15:2411)||2011|
|Not Applicable||Not Cited||
ON and OFF pathways in Drosophila motion vision.
A-11077 was used in immunohistochemistry to test how L1-L5 contribute to visual motion detection in Drosophila melanogaster.
|Joesch M,Schnell B,Raghu SV,Reiff DF,Borst A||Nature (468:300)||2010|
|Not Applicable||Not Cited||Monitoring autophagy in Alzheimer's disease and related neurodegenerative diseases.||Yang DS,Lee JH,Nixon RA||Methods in enzymology (453:111)||2009|
|Not Applicable||Not Cited||Angiogenesis selectively requires the p110alpha isoform of PI3K to control endothelial cell migration.||Graupera M,Guillermet-Guibert J,Foukas LC,Phng LK,Cain RJ,Salpekar A,Pearce W,Meek S,Millan J,Cutillas PR,Smith AJ,Ridley AJ,Ruhrberg C,Gerhardt H,Vanhaesebroeck B||Nature (453:662)||2008|
|Not Applicable||Not Cited||NKT cells are critical to initiate an inflammatory response after Pseudomonas aeruginosa ocular infection in susceptible mice.||Hazlett LD,Li Q,Liu J,McClellan S,Du W,Barrett RP||Journal of immunology (Baltimore, Md. : 1950) (179:1138)||2007|
|Not Applicable||Not Cited||Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology.||Singec I,Knoth R,Meyer RP,Maciaczyk J,Volk B,Nikkhah G,Frotscher M,Snyder EY||Nature methods (3:801)||2006|
|Not Applicable||Not Cited||Inhibition of platelet-derived growth factor-BB-induced receptor activation and fibroblast migration by hyaluronan activation of CD44.||Li L,Heldin CH,Heldin P||The Journal of biological chemistry (281:26512)||2006|
|Not Applicable||Not Cited||Mutant nuclear lamin A leads to progressive alterations of epigenetic control in premature aging.||Shumaker DK,Dechat T,Kohlmaier A,Adam SA,Bozovsky MR,Erdos MR,Eriksson M,Goldman AE,Khuon S,Collins FS,Jenuwein T,Goldman RD||Proceedings of the National Academy of Sciences of the United States of America (103:8703)||2006|
|Not Applicable||Not Cited||Fluoxetine targets early progenitor cells in the adult brain.||Encinas JM,Vaahtokari A,Enikolopov G||Proceedings of the National Academy of Sciences of the United States of America (103:8233)||2006|
|Not Applicable||Not Cited||Asynchronous nuclear division cycles in multinucleated cells.||Gladfelter AS,Hungerbuehler AK,Philippsen P||The Journal of cell biology (172:347)||2006|
|Not Applicable||Not Cited||Distinct chemokine triggers and in vivo migratory paths of fluorescein dye-labeled T Lymphocytes in acutely simian immunodeficiency virus SIVmac251-infected and uninfected macaques.||Clay CC,Rodrigues DS,Harvey DJ,Leutenegger CM,Esser U||Journal of virology (79:13759)||2005|
|Not Applicable||Not Cited||Glycosylation influences the lectin activities of the macrophage mannose receptor.||Su Y,Bakker T,Harris J,Tsang C,Brown GD,Wormald MR,Gordon S,Dwek RA,Rudd PM,Martinez-Pomares L||The Journal of biological chemistry (280:32811)||2005|
|Not Applicable||Not Cited||Phosphorylation of spinophilin by ERK and cyclin-dependent PK 5 (Cdk5).||Futter M,Uematsu K,Bullock SA,Kim Y,Hemmings HC,Nishi A,Greengard P,Nairn AC||Proceedings of the National Academy of Sciences of the United States of America (102:3489)||2005|
|Not Applicable||Not Cited||The C-type lectin receptor Endo180 displays internalization and recycling properties distinct from other members of the mannose receptor family.||Howard MJ,Isacke CM||The Journal of biological chemistry (277:32320)||2002|
|Not Applicable||Not Cited||Caveolin-1 and caveolin-2 expression in mouse macrophages. High density lipoprotein 3-stimulated secretion and a lack of significant subcellular co-localization.||Gargalovic P,Dory L||The Journal of biological chemistry (276:26164)||2001|
|Not Applicable||Not Cited||Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments.||Poupel O,Boleti H,Axisa S,Couture-Tosi E,Tardieux I||Molecular biology of the cell (11:355)||2000|