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HeLa cells were treated with 30 µM chloroquine and cultured overnight. The following day, the cells were fed 10 µM EdU under regular growth conditions for one hour and then fixed and permeabilized. EdU was used to was visualize proliferating cells using The Click-iT® EdU Alexa Fluor® 488 Imaging kit (pink). Cells were counter stained with 0.5 µg/mL anti-LC3B with a goat anti rabbit Alexa Fluor® 647 secondary (Green), mouse anti alpha tubulin with a goat anti mouse Alexa Fluor® 555 secondary (Cyan) and 1 µg/mL Hoechst 33342 (Blue). Cells were imaged on a Molecular Devices ImageXpress High content imager.
|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 bovine IgG, goat IgG, mouse IgG, rat IgG and human IgG|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|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-rabbit IgG whole antibodies have been cross-adsorbed against bovine IgG, goat IgG, mouse IgG, rat IgG, and human IgG. 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.
|Not Applicable||Not Cited||
Neuronal activity enhances tau propagation and tau pathology in vivo.
A-21245 was used in immunohistochemistry - free floating to characterize enhanced tau propagation and tau pathology in vivo by neuronal activity
|Wu JW,Hussaini SA,Bastille IM,Rodriguez GA,Mrejeru A,Rilett K,Sanders DW,Cook C,Fu H,Boonen RA,Herman M,Nahmani E,Emrani S,Figueroa YH,Diamond MI,Clelland CL,Wray S,Duff KE||Nature neuroscience (19:1085)||2016|
Subnuclear localization, rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield, confocal and super-resolution microscopy.
A-21245 was used in immunocytochemistry to study the effectiveness, rates, and subnuclear localization of UVC-induced unscheduled DNA synthesis visualized through fluorescence widefield, confocal and super-resolution microscopy
|Pierzy¿ska-Mach A,Szczurek A,Cella Zanacchi F,Pennacchietti F,Druka¿a J,Diaspro A,Cremer C,Darzynkiewicz Z,Dobrucki JW||Cell cycle (Georgetown, Tex.) (15:1156)||2016|
|Not Applicable||Not Cited||
Super-complexes of adhesion GPCRs and neural guidance receptors.
A-21245 was used in immunocytochemistry to analyze neural guidance receptors and super-complexes of adhesion GPCRs
|Jackson VA,Mehmood S,Chavent M,Roversi P,Carrasquero M,Del Toro D,Seyit-Bremer G,Ranaivoson FM,Comoletti D,Sansom MS,Robinson CV,Klein R,Seiradake E||Nature communications (7:null)||2016|
|Not Applicable||Not Cited||
Comparative transcriptomics reveals RhoE as a novel regulator of actin dynamics in bone-resorbing osteoclasts.
A-21245 was used in immunocytochemistry to study actin dynamics during podosome patterning.
|Georgess D,Mazzorana M,Terrado J,Delprat C,Chamot C,Guasch RM,Pérez-Roger I,Jurdic P,Machuca-Gayet I||Molecular biology of the cell (25:380)||2014|
An epigenetic feedback regulatory loop involving microRNA-195 and MBD1 governs neural stem cell differentiation.
A-21245 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-21245 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|
Nucleus accumbens D2R cells signal prior outcomes and control risky decision-making.
A-21245 was used in immunohistochemistry to assess the control for risky decision-making and signal for prior outcomes due to nucleus accumbens D2R cells signals
|Zalocusky KA,Ramakrishnan C,Lerner TN,Davidson TJ,Knutson B,Deisseroth K||Nature (531:642)||2016|
Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila.
A-21245 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|
Thymosin ß4-sulfoxide attenuates inflammatory cell infiltration and promotes cardiac wound healing.
A-21245 was used in immunohistochemistry to assess cardiac wound healing and inflammatory cell infiltration is attenuated by thymosin beta-4-sulfoxide
|Evans MA,Smart N,Dubé KN,Bollini S,Clark JE,Evans HG,Taams LS,Richardson R,Lévesque M,Martin P,Mills K,Riegler J,Price AN,Lythgoe MF,Riley PR||Nature communications (4:null)||2013|
|Not Applicable||Not Cited||Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging.||Szymborska A,de Marco A,Daigle N,Cordes VC,Briggs JA,Ellenberg J||Science (New York, N.Y.) (341:655)||2013|
|Not Applicable||Not Cited||A super-resolution map of the vertebrate kinetochore.||Ribeiro SA,Vagnarelli P,Dong Y,Hori T,McEwen BF,Fukagawa T,Flors C,Earnshaw WC||Proceedings of the National Academy of Sciences of the United States of America (107:10484)||2010|
|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||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||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||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||The cytosolic phospholipase A2 pathway, a safeguard of beta2-adrenergic cardiac effects in rat.||Ait-Mamar B,Cailleret M,Rucker-Martin C,Bouabdallah A,Candiani G,Adamy C,Duvaldestin P,Pecker F,Defer N,Pavoine C||The Journal of biological chemistry (280:18881)||2005|
|Not Applicable||Not Cited||Protein kinase Calpha activates c-Src and induces podosome formation via AFAP-110.||Gatesman A,Walker VG,Baisden JM,Weed SA,Flynn DC||Molecular and cellular biology (24:7578)||2004|
|Not Applicable||Not Cited||
Triggering the succinate receptor GPR91 on dendritic cells enhances immunity.
A-21245 was used in reverse phase protein array to investigate how the succinate receptor GPR91 contributes to immune responses
|Rubic T,Lametschwandtner G,Jost S,Hinteregger S,Kund J,Carballido-Perrig N,Schwärzler C,Junt T,Voshol H,Meingassner JG,Mao X,Werner G,Rot A,Carballido JM||Nature immunology (9:1261)||2008|