Immunofluorescence analysis of Donkey anti-Mouse IgG Secondary Antibody, Alexa Fluor 568 conjugate was performed using HeLa cells stained with alpha Tubulin (23610501) Mouse Monoclonal Primary Antibody (A11126). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, blocked with 1% BSA for 1 hour and labeled with 2ug/ml mouse primary antibody for 3 hours at room temperature. Donkey anti-Mouse IgG Secondary Antibody, Alexa Fluor 568 conjugate (A10037) was used at a concentration of 2ug/ml in phosphate buffered saline containing 0.2 % BSA for 45 minutes at room temperature, for detection of alpha Tubulin in the cytoplasm (Panel a: red). Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (S36938). F-actin was stained with Alexa Fluor® 488 Phalloidin (A12379, 1:300) (Panel c: green). Panel d represents the composite image. No nonspecific staining was observed with the secondary antibody alone (panel f), or with an isotype control (panel e). The images were captured at 60X magnification.
|Tested species reactivity||Mouse|
|Published species reactivity||Not Applicable|
|Host / Isotype||Donkey / IgG|
|Conjugate||Alexa Fluor® 568|
|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)||2 µg/ml|
|Immunofluorescence (IF)||2 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.
|Miscellaneous PubMed (MISC)||See 20 publications below|
These donkey anti-mouse IgG whole secondary antibodies have been affinity-purified and show minimum cross-reactivity to bovine, chicken, goat, guinea pig, hamster, horse, human, rabbit, 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 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.
|Not Applicable||Not Cited||
SCYL2 Protects CA3 Pyramidal Neurons from Excitotoxicity during Functional Maturation of the Mouse Hippocampus.
A10037 was used in immunohistochemistry - paraffin section to study the role of excitotoxicity in neuronal death
|Gingras S,Earls LR,Howell S,Smeyne RJ,Zakharenko SS,Pelletier S||The Journal of neuroscience : the official journal of the Society for Neuroscience (35:10510)||2015|
|Not Applicable||Not Cited||Calpain A modulates Toll responses by limited Cactus/I¿B proteolysis.||Fontenele M,Lim B,Oliveira D,Buffolo M,Perlman DH,Schupbach T,Araujo H||Molecular biology of the cell (24:2966)||2013|
|Not Applicable||Not Cited||Peroxiredoxin-2 represses melanoma metastasis by increasing E-Cadherin/ß-Catenin complexes in adherens junctions.||Lee DJ,Kang DH,Choi M,Choi YJ,Lee JY,Park JH,Park YJ,Lee KW,Kang SW||Cancer research (73:4744)||2013|
|Not Applicable||Not Cited||HDAC6 regulates mutant SOD1 aggregation through two SMIR motifs and tubulin acetylation.||Gal J,Chen J,Barnett KR,Yang L,Brumley E,Zhu H||The Journal of biological chemistry (288:15035)||2013|
|Not Applicable||Not Cited||Identification of a role for CLASP2 in insulin action.||Langlais P,Dillon JL,Mengos A,Baluch DP,Ardebili R,Miranda DN,Xie X,Heckmann BL,Liu J,Mandarino LJ||The Journal of biological chemistry (287:39245)||2012|
|Not Applicable||Not Cited||Membrane-associated aquaporin-1 facilitates osmotically driven water flux across the basolateral membrane of the thick ascending limb.||Cabral PD,Herrera M||American journal of physiology. Renal physiology (303:F621)||2012|
|Not Applicable||Not Cited||Extracellular Ca(2+) sensing in salivary ductal cells.||Bandyopadhyay BC,Swaim WD,Sarkar A,Liu X,Ambudkar IS||The Journal of biological chemistry (287:30305)||2012|
|Not Applicable||Not Cited||Passive immunoprotection of Plasmodium falciparum-infected mice designates the CyRPA as candidate malaria vaccine antigen.||Dreyer AM,Matile H,Papastogiannidis P,Kamber J,Favuzza P,Voss TS,Wittlin S,Pluschke G||Journal of immunology (Baltimore, Md. : 1950) (188:6225)||2012|
|Not Applicable||Not Cited||Cisplatin-induced ototoxicity is mediated by nitroxidative modification of cochlear proteins characterized by nitration of Lmo4.||Jamesdaniel S,Coling D,Hinduja S,Ding D,Li J,Cassidy L,Seigel GM,Qu J,Salvi R||The Journal of biological chemistry (287:18674)||2012|
|Not Applicable||Not Cited||Model for long QT syndrome type 2 using human iPS cells demonstrates arrhythmogenic characteristics in cell culture.||Lahti AL,Kujala VJ,Chapman H,Koivisto AP,Pekkanen-Mattila M,Kerkelä E,Hyttinen J,Kontula K,Swan H,Conklin BR,Yamanaka S,Silvennoinen O,Aalto-Setälä K||Disease models and mechanisms (5:220)||2012|
|Not Applicable||Not Cited||Lethal arrhythmias in Tbx3-deficient mice reveal extreme dosage sensitivity of cardiac conduction system function and homeostasis.||Frank DU,Carter KL,Thomas KR,Burr RM,Bakker ML,Coetzee WA,Tristani-Firouzi M,Bamshad MJ,Christoffels VM,Moon AM||Proceedings of the National Academy of Sciences of the United States of America (109:E154)||2012|
|Not Applicable||Not Cited||Cellular poly(c) binding proteins 1 and 2 interact with porcine reproductive and respiratory syndrome virus nonstructural protein 1ß and support viral replication.||Beura LK,Dinh PX,Osorio FA,Pattnaik AK||Journal of virology (85:12939)||2011|
|Not Applicable||Not Cited||Perinatal lipid nutrition alters early intestinal development and programs the response to experimental colitis in young adult rats.||Innis SM,Dai C,Wu X,Buchan AM,Jacobson K||American journal of physiology. Gastrointestinal and liver physiology (299:G1376)||2010|
|Not Applicable||Not Cited||Protein S protects neurons from excitotoxic injury by activating the TAM receptor Tyro3-phosphatidylinositol 3-kinase-Akt pathway through its sex hormone-binding globulin-like region.||Zhong Z,Wang Y,Guo H,Sagare A,Fernández JA,Bell RD,Barrett TM,Griffin JH,Freeman RS,Zlokovic BV||The Journal of neuroscience : the official journal of the Society for Neuroscience (30:15521)||2010|
|Not Applicable||Not Cited||Technical Advance: The rat aorta contains resident mononuclear phagocytes with proliferative capacity and proangiogenic properties.||Zorzi P,Aplin AC,Smith KD,Nicosia RF||Journal of leukocyte biology (88:1051)||2010|
|Not Applicable||Not Cited||Identification of RanBP 9/10 as interacting partners for protein kinase C (PKC) gamma/delta and the D1 dopamine receptor: regulation of PKC-mediated receptor phosphorylation.||Rex EB,Rankin ML,Yang Y,Lu Q,Gerfen CR,Jose PA,Sibley DR||Molecular pharmacology (78:69)||2010|
|Not Applicable||Not Cited||Platelet-associated complement factor H in healthy persons and patients with atypical HUS.||Licht C,Pluthero FG,Li L,Christensen H,Habbig S,Hoppe B,Geary DF,Zipfel PF,Kahr WH||Blood (114:4538)||2009|
|Not Applicable||Not Cited||Substance P (SP) enhances CCL5-induced chemotaxis and intracellular signaling in human monocytes, which express the truncated neurokinin-1 receptor (NK1R).||Chernova I,Lai JP,Li H,Schwartz L,Tuluc F,Korchak HM,Douglas SD,Kilpatrick LE||Journal of leukocyte biology (85:154)||2009|
|Not Applicable||Not Cited||The A- and B-type nuclear lamin networks: microdomains involved in chromatin organization and transcription.||Shimi T,Pfleghaar K,Kojima S,Pack CG,Solovei I,Goldman AE,Adam SA,Shumaker DK,Kinjo M,Cremer T,Goldman RD||Genes and development (22:3409)||2008|
|Not Applicable||Not Cited||Serum and glucocorticoid-regulated kinase Sgk1 inhibits insulin-dependent activation of phosphomannomutase 2 in transfected COS-7 cells.||Menniti M,Iuliano R,Amato R,Boito R,Corea M,Le Pera I,Gulletta E,Fuiano G,Perrotti N||American journal of physiology. Cell physiology (288:C148)||2005|