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Immunofluorescent detection of Zo-1 in MDCK cells. Confluent monolayers were fixed in 50%methanol/50%Acetone, blocked for at least 30 minutes in 1% BSA then incubated 2 hours with a Zo-1 rabbit polyclonal antibody (Product # 40-2200) at 2.5 ug/mL, washed, then incubated 1 hour with Alexa Flour 488 conjugated Donkey anti-Rabbit secondary antibody (Product # A21206) at 1:2000 dilution. Cells were counterstained with DAPI (blue). Coverslips were mounted with Prolong Gold Antifade reagent (Product # P36930) and imaged at 40X. Images generated by Joell Solan in Paul Lampe Lab at the Fred Hutchinson cancer Research Center.
|Tested species reactivity||Rabbit|
|Published species reactivity||Not Applicable|
|Host / Isotype||Donkey / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 488|
|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)||1-10 µg/mL|
|Immunocytochemistry (ICC)||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 minimum cross-reactivity to bovine, chicken, goat, guinea pig, hamster, horse, human, mouse, rat, and sheep serum proteins. 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 488 dye is a bright, green-fluorescent dye with excitation ideally suited to the 488 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 488 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 488 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||
STAM2, a member of the endosome-associated complex ESCRT-0 is highly expressed in neurons.
A-21206 was used in immunohistochemistry - frozen section to study STAM2 in the nervous system
|Kapuralin K,Ćurlin M,Mitrečić D,Kosi N,Schwarzer C,Glavan G,Gajović S||Molecular and cellular neurosciences (67:104)||2015|
|Not Applicable||Not Cited||
The methyl binding domain 3/nucleosome remodelling and deacetylase complex regulates neural cell fate determination and terminal differentiation in the cerebral cortex.
A-21206 was used in immunohistochemistry - frozen section to investigate the role of MBD3/NuRD in neurogenesis.
|Knock E,Pereira J,Lombard PD,Dimond A,Leaford D,Livesey FJ,Hendrich B||Neural development (10:null)||2015|
Ventral pallidal projections to mediodorsal thalamus and ventral tegmental area play distinct roles in outcome-specific Pavlovian-instrumental transfer.
A-21206 was used in immunohistochemistry - frozen section to study the Pavlovian-instrumental transfer effect of the rostral medial ventral pallidum region innervated by the nucleus accumbens shell
|Leung BK,Balleine BW||The Journal of neuroscience : the official journal of the Society for Neuroscience (35:4953)||2015|
Basonuclin-1 modulates epithelial plasticity and TGF-β1-induced loss of epithelial cell integrity.
A-21206 was used in immunocytochemistry to show that Basonuclin- regulates TGF-β-induced epithelial dedifferentiation of mammary epithelial cells.
|Feuerborn A,Mathow D,Srivastava PK,Gretz N,Gröne HJ||Oncogene (34:1185)||2015|
The nuclear form of glutathione peroxidase 4 colocalizes and directly interacts with protamines in the nuclear matrix during mouse sperm chromatin assembly.
A-21206 was used in immunocytochemistry to test if nGPx4 directly interacts with protamines by transiently sharing a nuclear matrix localization.
|Puglisi R,Maccari I,Pipolo S,Mangia F,Boitani C||Spermatogenesis (4:null)||2014|
HB-EGF affects astrocyte morphology, proliferation, differentiation, and the expression of intermediate filament proteins.
A-21206 was used in immunocytochemistry to report that HB-EGF-induced EGF receptor activation via phosphorylation and Mapk/Erk pathway activation results in increased cell proliferation.
|Puschmann TB,Zandén C,Lebkuechner I,Philippot C,de Pablo Y,Liu J,Pekny M||Journal of neurochemistry (128:878)||2014|
|Not Applicable||Not Cited||Interaction proteomics identify NEURL4 and the HECT E3 ligase HERC2 as novel modulators of centrosome architecture.||Al-Hakim AK,Bashkurov M,Gingras AC,Durocher D,Pelletier L||Molecular & cellular proteomics : MCP (11:null)||2012|
|Not Applicable||Not Cited||Contrasting roles of condensin I and condensin II in mitotic chromosome formation.||Green LC,Kalitsis P,Chang TM,Cipetic M,Kim JH,Marshall O,Turnbull L,Whitchurch CB,Vagnarelli P,Samejima K,Earnshaw WC,Choo KH,Hudson DF||Journal of cell science (125:1591)||2012|
|Not Applicable||Not Cited||Protease activated receptors 1 and 4 sensitize TRPV1 in nociceptive neurones.||Vellani V,Kinsey AM,Prandini M,Hechtfischer SC,Reeh P,Magherini PC,Giacomoni C,McNaughton PA||Molecular pain (6:null)||2010|
|Not Applicable||Not Cited||Immunohistochemical detection of soluble immunoglobulins in living mouse small intestines using an in vivo cryotechnique.||Shimo S,Saitoh S,Terada N,Ohno N,Saitoh Y,Ohno S||Journal of immunological methods (361:64)||2010|
|Not Applicable||Not Cited||A high-throughput, cell-based screening method for siRNA and small molecule inhibitors of mTORC1 signaling using the In Cell Western technique.||Hoffman GR,Moerke NJ,Hsia M,Shamu CE,Blenis J||Assay and drug development technologies (8:186)||2010|
|Not Applicable||Not Cited||Visualization and identification of IL-7 producing cells in reporter mice.||Mazzucchelli RI,Warming S,Lawrence SM,Ishii M,Abshari M,Washington AV,Feigenbaum L,Warner AC,Sims DJ,Li WQ,Hixon JA,Gray DH,Rich BE,Morrow M,Anver MR,Cherry J,Naf D,Sternberg LR,McVicar DW,Farr AG,Germain RN,Rogers K,Jenkins NA,Copeland NG,Durum SK||PloS one (4:null)||2009|
|Not Applicable||Not Cited||Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission.||Dagda RK,Cherra SJ,Kulich SM,Tandon A,Park D,Chu CT||The Journal of biological chemistry (284:13843)||2009|
|Not Applicable||Not Cited||Immunofluorescence imaging of DNA damage response proteins: optimizing protocols for super-resolution microscopy.||Bennett BT,Bewersdorf J,Knight KL||Methods (San Diego, Calif.) (48:63)||2009|
|Not Applicable||Not Cited||Unimpaired lysosomal acidification in respiratory epithelial cells in cystic fibrosis.||Haggie PM,Verkman AS||The Journal of biological chemistry (284:7681)||2009|
|Not Applicable||Not Cited||Site-specific modification of AAV vector particles with biophysical probes and targeting ligands using biotin ligase.||Stachler MD,Chen I,Ting AY,Bartlett JS||Molecular therapy : the journal of the American Society of Gene Therapy (16:1467)||2008|
|Not Applicable||Not Cited||DAG lipase activity is necessary for TRP channel regulation in Drosophila photoreceptors.||Leung HT,Tseng-Crank J,Kim E,Mahapatra C,Shino S,Zhou Y,An L,Doerge RW,Pak WL||Neuron (58:884)||2008|
|Not Applicable||Not Cited||Early resolution of acute immune activation and induction of PD-1 in SIV-infected sooty mangabeys distinguishes nonpathogenic from pathogenic infection in rhesus macaques.||Estes JD,Gordon SN,Zeng M,Chahroudi AM,Dunham RM,Staprans SI,Reilly CS,Silvestri G,Haase AT||Journal of immunology (Baltimore, Md. : 1950) (180:6798)||2008|
|Not Applicable||Not Cited||Characterization of rat rostral raphe primary cultures: multiplex quantification of serotonergic markers.||Czesak M,Burns AM,Remes Lenicov F,Albert PR||Journal of neuroscience methods (164:59)||2007|
|Not Applicable||Not Cited||Neutralization of human papillomavirus with monoclonal antibodies reveals different mechanisms of inhibition.||Day PM,Thompson CD,Buck CB,Pang YY,Lowy DR,Schiller JT||Journal of virology (81:8784)||2007|
|Not Applicable||Not Cited||The induced prostaglandin E2 pathway is a key regulator of the respiratory response to infection and hypoxia in neonates.||Hofstetter AO,Saha S,Siljehav V,Jakobsson PJ,Herlenius E||Proceedings of the National Academy of Sciences of the United States of America (104:9894)||2007|
|Not Applicable||Not Cited||Specialized inhibitory synaptic actions between nearby neocortical pyramidal neurons.||Ren M,Yoshimura Y,Takada N,Horibe S,Komatsu Y||Science (New York, N.Y.) (316:758)||2007|
|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||Identification of mouse Prp19p as a lipid droplet-associated protein and its possible involvement in the biogenesis of lipid droplets.||Cho SY,Shin ES,Park PJ,Shin DW,Chang HK,Kim D,Lee HH,Lee JH,Kim SH,Song MJ,Chang IS,Lee OS,Lee TR||The Journal of biological chemistry (282:2456)||2007|
|Not Applicable||Not Cited||Visualization in zebrafish larvae of Na(+) uptake in mitochondria-rich cells whose differentiation is dependent on foxi3a.||Esaki M,Hoshijima K,Kobayashi S,Fukuda H,Kawakami K,Hirose S||American journal of physiology. Regulatory, integrative and comparative physiology (292:R470)||2007|
|Not Applicable||Not Cited||Chromatin-remodeling factors allow differentiation of bone marrow cells into insulin-producing cells.||Thatava T,Tayaramma T,Ma B,Rohde M,Mayer H||Stem cells (Dayton, Ohio) (24:2858)||2006|
|Not Applicable||Not Cited||Destruction of pancreatic beta-cells by transgenic induction of prostaglandin E2 in the islets.||Oshima H,Taketo MM,Oshima M||The Journal of biological chemistry (281:29330)||2006|
|Not Applicable||Not Cited||Differential expression of cell surface markers by ovine respiratory tract dendritic cells.||McNeilly TN,Brown JK,Harkiss G||The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society (54:1021)||2006|
|Not Applicable||Not Cited||Saccharomyces cerevisiae Hog1 protein phosphorylation upon exposure to bacterial endotoxin.||Marques JM,Rodrigues RJ,de Magalhães-Sant'ana AC,Gonçalves T||The Journal of biological chemistry (281:24687)||2006|
|Not Applicable||Not Cited||Endoplasmic reticulum stress associated with extracellular aggregates. Evidence from transthyretin deposition in familial amyloid polyneuropathy.||Teixeira PF,Cerca F,Santos SD,Saraiva MJ||The Journal of biological chemistry (281:21998)||2006|
|Not Applicable||Not Cited||Characterization of leptin-responsive neurons in the caudal brainstem.||Ellacott KL,Halatchev IG,Cone RD||Endocrinology (147:3190)||2006|
|Not Applicable||Not Cited||Zinc transport complexes contribute to the homeostatic maintenance of secretory pathway function in vertebrate cells.||Ishihara K,Yamazaki T,Ishida Y,Suzuki T,Oda K,Nagao M,Yamaguchi-Iwai Y,Kambe T||The Journal of biological chemistry (281:17743)||2006|
|Not Applicable||Not Cited||Tbx1 haploinsufficiency is linked to behavioral disorders in mice and humans: implications for 22q11 deletion syndrome.||Paylor R,Glaser B,Mupo A,Ataliotis P,Spencer C,Sobotka A,Sparks C,Choi CH,Oghalai J,Curran S,Murphy KC,Monks S,Williams N,O'Donovan MC,Owen MJ,Scambler PJ,Lindsay E||Proceedings of the National Academy of Sciences of the United States of America (103:7729)||2006|
|Not Applicable||Not Cited||The adaptor protein Nck interacts with Fas ligand: Guiding the death factor to the cytotoxic immunological synapse.||Lettau M,Qian J,Linkermann A,Latreille M,Larose L,Kabelitz D,Janssen O||Proceedings of the National Academy of Sciences of the United States of America (103:5911)||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||A novel mechanism for protein delivery: granzyme B undergoes electrostatic exchange from serglycin to target cells.||Raja SM,Metkar SS,Höning S,Wang B,Russin WA,Pipalia NH,Menaa C,Belting M,Cao X,Dressel R,Froelich CJ||The Journal of biological chemistry (280:20752)||2005|
|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|
|Not Applicable||Not Cited||A novel specific role for I kappa B kinase complex-associated protein in cytosolic stress signaling.||Holmberg C,Katz S,Lerdrup M,Herdegen T,Jäättelä M,Aronheim A,Kallunki T||The Journal of biological chemistry (277:31918)||2002|
|Not Applicable||Not Cited||Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.||Howell BJ,McEwen BF,Canman JC,Hoffman DB,Farrar EM,Rieder CL,Salmon ED||The Journal of cell biology (155:1159)||2001|
|Not Applicable||Not Cited||Requirement for N-ethylmaleimide-sensitive factor activity at different stages of bacterial invasion and phagocytosis.||Coppolino MG,Kong C,Mohtashami M,Schreiber AD,Brumell JH,Finlay BB,Grinstein S,Trimble WS||The Journal of biological chemistry (276:4772)||2001|