Immunofluorescence analysis of Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 633 conjugate was performed using MCF-7 cells stained with Cytokeratin 19 (RCK108) Mouse Monoclonal Primary Antibody (MA512613). 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 Mouse primary antibody (1:250 dilution) for 3 hours at room temperature. Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 633 conjugate (A21052) was used at a concentration of 4µg/ml in phosphate buffered saline containing 0.2 % BSA for 45 minutes at room temperature, for detection of Cytokeratin 19 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||Goat / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 633|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Cross Adsorption||Against bovine IgG, goat IgG, rabbit IgG, rat IgG, human IgG and human serum|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1-10 µg/mL|
|Immunocytochemistry (ICC)||4 µg/ml|
|Immunofluorescence (IF)||4 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-mouse IgG (H+L) whole secondary antibodies have been affinity purified and cross-adsorbed against bovine IgG, goat IgG, rabbit IgG, rat IgG, human IgG, and human 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 633 dye is a bright, far-red-fluorescent dye with excitation ideally suited to the 633 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 633 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 633 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||
PINK1 deficiency enhances autophagy and mitophagy induction.
A-21052 was used in immunocytochemistry to study enhanced mitophagy and autophagy induction by PINK1 deficiency
|Gómez-Sánchez R,Yakhine-Diop SM,Bravo-San Pedro JM,Pizarro-Estrella E,Rodríguez-Arribas M,Climent V,Martin-Cano FE,González-Soltero ME,Tandon A,Fuentes JM,González-Polo RA||Molecular and cellular oncology (3:null)||2016|
HACE1-dependent protein degradation provides cardiac protection in response to haemodynamic stress.
A-21052 was used in immunocytochemistry to demonstrate that HACE protects the heart under pressure stress by regulating protein degradation.
|Zhang L,Chen X,Sharma P,Moon M,Sheftel AD,Dawood F,Nghiem MP,Wu J,Li RK,Gramolini AO,Sorensen PH,Penninger JM,Brumell JH,Liu PP||Nature communications (5:null)||2014|
Adult-born and preexisting olfactory granule neurons undergo distinct experience-dependent modifications of their olfactory responses in vivo.
A-21052 was used in immunocytochemistry to test if recently generated, adult-born neurons contribute to neural plasticity and learning
|Magavi SS,Mitchell BD,Szentirmai O,Carter BS,Macklis JD||The Journal of neuroscience : the official journal of the Society for Neuroscience (25:10729)||2005|
Gene therapy restores vision in rd1 mice after removal of a confounding mutation in Gpr179.
A-21052 was used in immunohistochemistry - frozen section to investigate how the Grp179 mutation contributes to bipolar cell-mediated b-waves in mice.
|Nishiguchi KM,Carvalho LS,Rizzi M,Powell K,Holthaus SM,Azam SA,Duran Y,Ribeiro J,Luhmann UF,Bainbridge JW,Smith AJ,Ali RR||Nature communications (6:null)||2015|
Physiological and stem cell compartmentalization within the Drosophila midgut.
A-21052 was used in immunohistochemistry to compare intestinal stem cells from different subregions
|Marianes A,Spradling AC||eLife (2:null)||2013|
|Not Applicable||Not Cited||
Querkopf is a key marker of self-renewal and multipotency of adult neural stem cells.
A-21052 was used in immunohistochemistry to elucidate the factors regulating adult neural stem cells in vivo
|Sheikh BN,Dixon MP,Thomas T,Voss AK||Journal of cell science (125:295)||2012|
|Not Applicable||Not Cited||Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.||Narendra D,Tanaka A,Suen DF,Youle RJ||The Journal of cell biology (183:795)||2008|
|Not Applicable||Not Cited||The impact of different nanoparticle surface chemistry and size on uptake and toxicity in a murine macrophage cell line.||Clift MJ,Rothen-Rutishauser B,Brown DM,Duffin R,Donaldson K,Proudfoot L,Guy K,Stone V||Toxicology and applied pharmacology (232:418)||2008|
|Not Applicable||Not Cited||Time-lapse analysis and mathematical characterization elucidate novel mechanisms underlying muscle morphogenesis.||Snow CJ,Goody M,Kelly MW,Oster EC,Jones R,Khalil A,Henry CA||PLoS genetics (4:null)||2008|
|Not Applicable||Not Cited||TLR ligand-induced podosome disassembly in dendritic cells is ADAM17 dependent.||West MA,Prescott AR,Chan KM,Zhou Z,Rose-John S,Scheller J,Watts C||The Journal of cell biology (182:993)||2008|
|Not Applicable||Not Cited||Quantum dot-induced epigenetic and genotoxic changes in human breast cancer cells.||Choi AO,Brown SE,Szyf M,Maysinger D||Journal of molecular medicine (Berlin, Germany) (86:291)||2008|
|Not Applicable||Not Cited||Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.||Sakaue-Sawano A,Kurokawa H,Morimura T,Hanyu A,Hama H,Osawa H,Kashiwagi S,Fukami K,Miyata T,Miyoshi H,Imamura T,Ogawa M,Masai H,Miyawaki A||Cell (132:487)||2008|
|Not Applicable||Not Cited||Apoptosis in tumour cells photosensitized with Rose Bengal acetate is induced by multiple organelle photodamage.||Soldani C,Croce AC,Bottone MG,Fraschini A,Biggiogera M,Bottiroli G,Pellicciari C||Histochemistry and cell biology (128:485)||2007|
|Not Applicable||Not Cited||Close relation of arterial ICC-like cells to the contractile phenotype of vascular smooth muscle cell.||Pucovský V,Harhun MI,Povstyan OV,Gordienko DV,Moss RF,Bolton TB||Journal of cellular and molecular medicine (11:764)||2007|
|Not Applicable||Not Cited||One-step analysis of protein complexes in microliters of cell lysate using indirect immunolabeling and fluorescence cross-correlation spectroscopy.||Stoevesandt O,Brock R||Nature protocols (1:223)||2007|
|Not Applicable||Not Cited||Internalization of novel non-viral vector TAT-streptavidin into human cells.||Rinne J,Albarran B,Jylhävä J,Ihalainen TO,Kankaanpää P,Hytönen VP,Stayton PS,Kulomaa MS,Vihinen-Ranta M||BMC biotechnology (7:null)||2007|
|Not Applicable||Not Cited||Molecular dynamics of a presynaptic active zone protein studied in Munc13-1-enhanced yellow fluorescent protein knock-in mutant mice.||Kalla S,Stern M,Basu J,Varoqueaux F,Reim K,Rosenmund C,Ziv NE,Brose N||The Journal of neuroscience : the official journal of the Society for Neuroscience (26:13054)||2006|
|Not Applicable||Not Cited||The P2Y12 receptor regulates microglial activation by extracellular nucleotides.||Haynes SE,Hollopeter G,Yang G,Kurpius D,Dailey ME,Gan WB,Julius D||Nature neuroscience (9:1512)||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||Prostate-derived sterile 20-like kinase 2 (PSK2) regulates apoptotic morphology via C-Jun N-terminal kinase and Rho kinase-1.||Zihni C,Mitsopoulos C,Tavares IA,Ridley AJ,Morris JD||The Journal of biological chemistry (281:7317)||2006|
|Not Applicable||Not Cited||Spatially organised mitochondrial calcium uptake through a novel pathway in chick neurones.||Coatesworth W,Bolsover S||Cell calcium (39:217)||2006|
|Not Applicable||Not Cited||Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking.||Arniges M,Fernández-Fernández JM,Albrecht N,Schaefer M,Valverde MA||The Journal of biological chemistry (281:1580)||2006|
|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||Stem cell division is regulated by the microRNA pathway.||Hatfield SD,Shcherbata HR,Fischer KA,Nakahara K,Carthew RW,Ruohola-Baker H||Nature (435:974)||2005|
|Not Applicable||Not Cited||Nuclear transport of single molecules: dwell times at the nuclear pore complex.||Kubitscheck U,Grünwald D,Hoekstra A,Rohleder D,Kues T,Siebrasse JP,Peters R||The Journal of cell biology (168:233)||2005|
|Not Applicable||Not Cited||Precise localization of alpha7 nicotinic acetylcholine receptors on glutamatergic axon terminals in the rat ventral tegmental area.||Jones IW,Wonnacott S||The Journal of neuroscience : the official journal of the Society for Neuroscience (24:11244)||2004|
|Not Applicable||Not Cited||Espins are multifunctional actin cytoskeletal regulatory proteins in the microvilli of chemosensory and mechanosensory cells.||Sekerková G,Zheng L,Loomis PA,Changyaleket B,Whitlon DS,Mugnaini E,Bartles JR||The Journal of neuroscience : the official journal of the Society for Neuroscience (24:5445)||2004|
|Not Applicable||Not Cited||Transformation of olfactory representations in the Drosophila antennal lobe.||Wilson RI,Turner GC,Laurent G||Science (New York, N.Y.) (303:366)||2004|
|Not Applicable||Not Cited||Reduced cell surface expression of CCR5 in CCR5Delta 32 heterozygotes is mediated by gene dosage, rather than by receptor sequestration.||Venkatesan S,Petrovic A,Van Ryk DI,Locati M,Weissman D,Murphy PM||The Journal of biological chemistry (277:2287)||2002|
|Not Applicable||Not Cited||Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae.||Mironov AA,Beznoussenko GV,Nicoziani P,Martella O,Trucco A,Kweon HS,Di Giandomenico D,Polishchuk RS,Fusella A,Lupetti P,Berger EG,Geerts WJ,Koster AJ,Burger KN,Luini A||The Journal of cell biology (155:1225)||2001|