Acetylcholine receptors were stained with the tetramethylrhodamine conjugate of a-bungarotoxin (Cat. No. T1175). Axons were labeled with a primary antibody against neurofilaments and visualized with a green-fluorescent, fluorescein-labeled secondary antibody. Myelin was labeled using an antibody against P0 protein, visualized with Alexa Fluor® 647 goat anti–mouse IgG antibody (Cat. No. A21244) and pseudocolored blue. Image contributed by Felipe Court and R.R. Ribchester, University of Edinburgh, Scotland.
|Tested species reactivity||Rabbit|
|Published species reactivity||Not Applicable|
|Host / Isotype||Goat / 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|
|Cross Adsorption||Against human IgG, human serum, mouse IgG, mouse serum and bovine serum|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1-10 µg/mL|
|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.
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 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.
Inducible depletion of adult skeletal muscle stem cells impairs the regeneration of neuromuscular junctions.
A-21244 was used in immunohistochemistry to analyze impairment of the regeneration of neuromuscular junctions by an inducible depletion of adult skeletal muscle stem cells
|Liu W,Wei-LaPierre L,Klose A,Dirksen RT,Chakkalakal JV||eLife (4:null)||2015|
High-performance probes for light and electron microscopy.
A-21244 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|
Fibrillar amyloid plaque formation precedes microglial activation.
A-21244 was used in immunohistochemistry to study the interactions of beta-amyloid deposits and microglia in a mouse model of Alzheimer's disease
|Jung CK,Keppler K,Steinbach S,Blazquez-Llorca L,Herms J||PloS one (10:null)||2015|
Arteries are formed by vein-derived endothelial tip cells.
A-21244 was used in immunohistochemistry to report that vein-derived endothelial tip cells contribute to emerging arteries in zebrafish
|Xu C,Hasan SS,Schmidt I,Rocha SF,Pitulescu ME,Bussmann J,Meyen D,Raz E,Adams RH,Siekmann AF||Nature communications (5:null)||2014|
|Not Applicable||20 µg/ml||
Discontinuous LYVE-1 expression in corneal limbal lymphatics: dual function as microvalves and immunological hot spots.
A-21244 was used in immunohistochemistry to examine LYVE-1 expression in corneal lymphatics.
|Nakao S,Zandi S,Faez S,Kohno R,Hafezi-Moghadam A||FASEB journal : official publication of the Federation of American Societies for Experimental Biology (26:808)||2012|
|Not Applicable||Not Cited||Promoter- and RNA polymerase II-dependent hsp-16 gene association with nuclear pores in Caenorhabditis elegans.||Rohner S,Kalck V,Wang X,Ikegami K,Lieb JD,Gasser SM,Meister P||The Journal of cell biology (200:589)||2013|
|Not Applicable||Not Cited||LY2228820 dimesylate, a selective inhibitor of p38 mitogen-activated protein kinase, reduces angiogenic endothelial cord formation in vitro and in vivo.||Tate CM,Blosser W,Wyss L,Evans G,Xue Q,Pan Y,Stancato L||The Journal of biological chemistry (288:6743)||2013|
|Not Applicable||Not Cited||Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules.||Burnette DT,Sengupta P,Dai Y,Lippincott-Schwartz J,Kachar B||Proceedings of the National Academy of Sciences of the United States of America (108:21081)||2011|
|Not Applicable||Not Cited||Combined genomic and phenotype screening reveals secretory factor SPINK1 as an invasion and survival factor associated with patient prognosis in breast cancer.||Soon WW,Miller LD,Black MA,Dalmasso C,Chan XB,Pang B,Ong CW,Salto-Tellez M,Desai KV,Liu ET||EMBO molecular medicine (3:451)||2011|
|Not Applicable||Not Cited||High-content, high-throughput analysis of cell cycle perturbations induced by the HSP90 inhibitor XL888.||Lyman SK,Crawley SC,Gong R,Adamkewicz JI,McGrath G,Chew JY,Choi J,Holst CR,Goon LH,Detmer SA,Vaclavikova J,Gerritsen ME,Blake RA||PloS one (6:null)||2011|
|Not Applicable||Not Cited||Surfactant protein A (SP-A)-mediated clearance of Staphylococcus aureus involves binding of SP-A to the staphylococcal adhesin eap and the macrophage receptors SP-A receptor 210 and scavenger receptor class A.||Sever-Chroneos Z,Krupa A,Davis J,Hasan M,Yang CH,Szeliga J,Herrmann M,Hussain M,Geisbrecht BV,Kobzik L,Chroneos ZC||The Journal of biological chemistry (286:4854)||2011|
|Not Applicable||Not Cited||Features of a spatially constrained cystine loop in the p10 FAST protein ectodomain define a new class of viral fusion peptides.||Barry C,Key T,Haddad R,Duncan R||The Journal of biological chemistry (285:16424)||2010|
|Not Applicable||Not Cited||Restriction of receptor movement alters cellular response: physical force sensing by EphA2.||Salaita K,Nair PM,Petit RS,Neve RM,Das D,Gray JW,Groves JT||Science (New York, N.Y.) (327:1380)||2010|
|Not Applicable||Not Cited||Robust fluorescent detection of protein fatty-acylation with chemical reporters.||Charron G,Zhang MM,Yount JS,Wilson J,Raghavan AS,Shamir E,Hang HC||Journal of the American Chemical Society (131:4967)||2009|
|Not Applicable||Not Cited||Endosomal trafficking of the ligated FcvarepsilonRI receptor.||Fattakhova GV,Masilamani M,Narayanan S,Borrego F,Gilfillan AM,Metcalfe DD,Coligan JE||Molecular immunology (46:793)||2009|
|Not Applicable||Not Cited||Involvement of mitochondrial signaling pathways in the mechanism of Fas-mediated apoptosis after spinal cord injury.||Yu WR,Liu T,Fehlings TK,Fehlings MG||The European journal of neuroscience (29:114)||2009|
|Not Applicable||Not Cited||Identification of a nonkinase target mediating cytotoxicity of novel kinase inhibitors.||Ross-Macdonald P,de Silva H,Guo Q,Xiao H,Hung CY,Penhallow B,Markwalder J,He L,Attar RM,Lin TA,Seitz S,Tilford C,Wardwell-Swanson J,Jackson D||Molecular cancer therapeutics (7:3490)||2008|
|Not Applicable||Not Cited||The transmembrane domain of the severe acute respiratory syndrome coronavirus ORF7b protein is necessary and sufficient for its retention in the Golgi complex.||Schaecher SR,Diamond MS,Pekosz A||Journal of virology (82:9477)||2008|
|Not Applicable||Not Cited||Niemann-Pick C1 functions in regulating lysosomal amine content.||Kaufmann AM,Krise JP||The Journal of biological chemistry (283:24584)||2008|
|Not Applicable||Not Cited||Endophilin B1 as a novel regulator of nerve growth factor/ TrkA trafficking and neurite outgrowth.||Wan J,Cheung AY,Fu WY,Wu C,Zhang M,Mobley WC,Cheung ZH,Ip NY||The Journal of neuroscience : the official journal of the Society for Neuroscience (28:9002)||2008|
|Not Applicable||Not Cited||The NALP3 inflammasome is involved in the innate immune response to amyloid-beta.||Halle A,Hornung V,Petzold GC,Stewart CR,Monks BG,Reinheckel T,Fitzgerald KA,Latz E,Moore KJ,Golenbock DT||Nature immunology (9:857)||2008|
|Not Applicable||Not Cited||Dicer inactivation leads to progressive functional and structural degeneration of the mouse retina.||Damiani D,Alexander JJ,O'Rourke JR,McManus M,Jadhav AP,Cepko CL,Hauswirth WW,Harfe BD,Strettoi E||The Journal of neuroscience : the official journal of the Society for Neuroscience (28:4878)||2008|
|Not Applicable||Not Cited||Caveolin regulates endocytosis of the muscle repair protein, dysferlin.||Hernández-Deviez DJ,Howes MT,Laval SH,Bushby K,Hancock JF,Parton RG||The Journal of biological chemistry (283:6476)||2008|
|Not Applicable||Not Cited||Invasive Escherichia coli are a feature of Crohn's disease.||Sasaki M,Sitaraman SV,Babbin BA,Gerner-Smidt P,Ribot EM,Garrett N,Alpern JA,Akyildiz A,Theiss AL,Nusrat A,Klapproth JM||Laboratory investigation; a journal of technical methods and pathology (87:1042)||2007|
|Not Applicable||Not Cited||High-content imaging analysis of the knockdown effects of validated siRNAs and antisense oligonucleotides.||Low J,Dowless M,Blosser W,Vincent T,Davis S,Hodson J,Koller E,Marcusson E,Blanchard K,Stancato L||Journal of biomolecular screening (12:775)||2007|
|Not Applicable||Not Cited||Isolation, characterization, and differentiation to hepatocyte-like cells of nonparenchymal epithelial cells from adult human liver.||Duret C,Gerbal-Chaloin S,Ramos J,Fabre JM,Jacquet E,Navarro F,Blanc P,Sa-Cunha A,Maurel P,Daujat-Chavanieu M||Stem cells (Dayton, Ohio) (25:1779)||2007|
|Not Applicable||Not Cited||Distribution of protein A on the surface of Staphylococcus aureus.||DeDent AC,McAdow M,Schneewind O||Journal of bacteriology (189:4473)||2007|
|Not Applicable||Not Cited||Diffusional trapping of GluR1 AMPA receptors by input-specific synaptic activity.||Ehlers MD,Heine M,Groc L,Lee MC,Choquet D||Neuron (54:447)||2007|
|Not Applicable||Not Cited||In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2.||Arenkiel BR,Peca J,Davison IG,Feliciano C,Deisseroth K,Augustine GJ,Ehlers MD,Feng G||Neuron (54:205)||2007|
|Not Applicable||Not Cited||Asymmetric T lymphocyte division in the initiation of adaptive immune responses.||Chang JT,Palanivel VR,Kinjyo I,Schambach F,Intlekofer AM,Banerjee A,Longworth SA,Vinup KE,Mrass P,Oliaro J,Killeen N,Orange JS,Russell SM,Weninger W,Reiner SL||Science (New York, N.Y.) (315:1687)||2007|
|Not Applicable||Not Cited||The contribution of bone marrow-derived cells to the development of renal interstitial fibrosis.||Li J,Deane JA,Campanale NV,Bertram JF,Ricardo SD||Stem cells (Dayton, Ohio) (25:697)||2007|
|Not Applicable||Not Cited||Self-renewing and differentiating properties of cortical neural stem cells are selectively regulated by basic fibroblast growth factor (FGF) signaling via specific FGF receptors.||Maric D,Fiorio Pla A,Chang YH,Barker JL||The Journal of neuroscience : the official journal of the Society for Neuroscience (27:1836)||2007|
|Not Applicable||Not Cited||Gamma-adaptin, a novel ubiquitin-interacting adaptor, and Nedd4 ubiquitin ligase control hepatitis B virus maturation.||Rost M,Mann S,Lambert C,Döring T,Thomé N,Prange R||The Journal of biological chemistry (281:29297)||2006|
|Not Applicable||Not Cited||Pregnane X receptor (PXR) activation: a mechanism for neuroprotection in a mouse model of Niemann-Pick C disease.||Langmade SJ,Gale SE,Frolov A,Mohri I,Suzuki K,Mellon SH,Walkley SU,Covey DF,Schaffer JE,Ory DS||Proceedings of the National Academy of Sciences of the United States of America (103:13807)||2006|
|Not Applicable||Not Cited||Microfluidic analysis of antibody specificity in a compact disk format.||Eriksson C,Agaton C,Kånge R,Sundberg M,Nilsson P,Ek B,Uhlén M,Gustafsson M,Hober S||Journal of proteome research (5:1568)||2006|
|Not Applicable||Not Cited||A macroporous hydrogel for the coculture of neural progenitor and endothelial cells to form functional vascular networks in vivo.||Ford MC,Bertram JP,Hynes SR,Michaud M,Li Q,Young M,Segal SS,Madri JA,Lavik EB||Proceedings of the National Academy of Sciences of the United States of America (103:2512)||2006|
|Not Applicable||Not Cited||Direct production and purification of T7 phage display cloned proteins selected and analyzed on microarrays.||Nowak JE,Chatterjee M,Mohapatra S,Dryden SC,Tainsky MA||BioTechniques (40:220)||2006|
|Not Applicable||Not Cited||The identification and characterization of two phosphatidylinositol-4,5-bisphosphate 4-phosphatases.||Ungewickell A,Hugge C,Kisseleva M,Chang SC,Zou J,Feng Y,Galyov EE,Wilson M,Majerus PW||Proceedings of the National Academy of Sciences of the United States of America (102:18854)||2005|
|Not Applicable||Not Cited||The anti-apoptotic protein Mcl-1 inhibits mitochondrial Ca2+ signals.||Minagawa N,Kruglov EA,Dranoff JA,Robert ME,Gores GJ,Nathanson MH||The Journal of biological chemistry (280:33637)||2005|
|Not Applicable||Not Cited||Receptor (CD155)-dependent endocytosis of poliovirus and retrograde axonal transport of the endosome.||Ohka S,Matsuda N,Tohyama K,Oda T,Morikawa M,Kuge S,Nomoto A||Journal of virology (78:7186)||2004|
|Not Applicable||Not Cited||Localization of cystic fibrosis transmembrane conductance regulator to lipid rafts of epithelial cells is required for Pseudomonas aeruginosa-induced cellular activation.||Kowalski MP,Pier GB||Journal of immunology (Baltimore, Md. : 1950) (172:418)||2004|
|Not Applicable||Not Cited||Caspase activation contributes to delayed death of heat-stressed striatal neurons.||White MG,Emery M,Nonner D,Barrett JN||Journal of neurochemistry (87:958)||2003|
|Not Applicable||Not Cited||A novel approach to in situ characterization of pancreatic beta-cells.||Speier S,Rupnik M||Pflugers Archiv : European journal of physiology (446:553)||2003|
|Not Applicable||Not Cited||HIV-1 Nef stabilizes the association of adaptor protein complexes with membranes.||Janvier K,Craig H,Hitchin D,Madrid R,Sol-Foulon N,Renault L,Cherfils J,Cassel D,Benichou S,Guatelli J||The Journal of biological chemistry (278:8725)||2003|
|Not Applicable||Not Cited||
The novel, orally bioavailable HSP90 inhibitor NVP-HSP990 induces cell cycle arrest and apoptosis in multiple myeloma cells and acts synergistically with melphalan by increased cleavage of caspases.
A-21244 was used in flow cytometry to analyze the effects of a HSP90 inhibitor, NVP-HSP990, on multiple myeloma cell proliferation and survival
|Lamottke B,Kaiser M,Mieth M,Heider U,Gao Z,Nikolova Z,Jensen MR,Sterz J,von Metzler I,Sezer O||European journal of haematology (88:406)||2012|
|Not Applicable||Not Cited||
Interleukin-1beta and tumor necrosis factor-alpha are expressed by different subsets of microglia and macrophages after ischemic stroke in mice.
A-21244 was used in flow cytometry 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|