Tetrahymena pyriformis were cultured with EdU (A10044), Click-iT® GalNAz glycoprotein labeling reagent (C33365), and InSpeck™ Blue (350/440) Intensity Calibration microspheres (I7221). Following fix/perm using Image-iT™ Fixation/Permeabilization kit (R37602), EdU-incorporated DNA was labeled with Alexa Fluor® 488 azide (A10266) and GalNAz-incorporated cellular components with Alexa Fluor® 555 alkyne (A20013). Cilia were labeled with anti-beta-tubulin Ab (32-2600) and Alexa Fluor® 647 secondary Ab (A21236). Imaging followed mounting in SlowFade® Gold (S36937).
|Tested species reactivity||Mouse|
|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, 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)||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-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 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||
A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation.
A-21236 was used in immunocytochemistry to characterize mammalian nuclear RNA N6-adenosine methylation mediation by a METTL3-METTL14 complex
|Liu J,Yue Y,Han D,Wang X,Fu Y,Zhang L,Jia G,Yu M,Lu Z,Deng X,Dai Q,Chen W,He C||Nature chemical biology (10:93)||2014|
N6-methyladenosine-dependent regulation of messenger RNA stability.
A-21236 was used in immunocytochemistry to elucidate the function of m(6)A modifications.
|Wang X,Lu Z,Gomez A,Hon GC,Yue Y,Han D,Fu Y,Parisien M,Dai Q,Jia G,Ren B,Pan T,He C||Nature (505:117)||2014|
|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||Systems survey of endocytosis by multiparametric image analysis.||Collinet C,Stöter M,Bradshaw CR,Samusik N,Rink JC,Kenski D,Habermann B,Buchholz F,Henschel R,Mueller MS,Nagel WE,Fava E,Kalaidzidis Y,Zerial M||Nature (464:243)||2010|
|Not Applicable||Not Cited||Opposite effects of simvastatin on the bactericidal and inflammatory response of macrophages to opsonized S. aureus.||Benati D,Ferro M,Savino MT,Ulivieri C,Schiavo E,Nuccitelli A,Pasini FL,Baldari CT||Journal of leukocyte biology (87:433)||2010|
|Not Applicable||Not Cited||A protein microarray-based analysis of S-nitrosylation.||Foster MW,Forrester MT,Stamler JS||Proceedings of the National Academy of Sciences of the United States of America (106:18948)||2009|
|Not Applicable||Not Cited||Sortase A localizes to distinct foci on the Streptococcus pyogenes membrane.||Raz A,Fischetti VA||Proceedings of the National Academy of Sciences of the United States of America (105:18549)||2008|
|Not Applicable||Not Cited||Profiling antibody responses by multiparametric analysis of primary B cells.||Story CM,Papa E,Hu CC,Ronan JL,Herlihy K,Ploegh HL,Love JC||Proceedings of the National Academy of Sciences of the United States of America (105:17902)||2008|
|Not Applicable||Not Cited||Assessment of golgi apparatus versus plasma membrane-localized multi-drug resistance-associated protein 1.||Kaufmann AM,Toro-Ramos AJ,Krise JP||Molecular pharmaceutics (5:787)||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||Nicotinic acetylcholine receptor is internalized via a Rac-dependent, dynamin-independent endocytic pathway.||Kumari S,Borroni V,Chaudhry A,Chanda B,Massol R,Mayor S,Barrantes FJ||The Journal of cell biology (181:1179)||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||A FRET-based fluorogenic phosphine for live-cell imaging with the Staudinger ligation.||Hangauer MJ,Bertozzi CR||Angewandte Chemie (International ed. in English) (47:2394)||2008|
|Not Applicable||Not Cited||Resolution of de novo HIV production and trafficking in immature dendritic cells.||Turville SG,Aravantinou M,Stössel H,Romani N,Robbiani M||Nature methods (5:75)||2008|
|Not Applicable||Not Cited||Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye.||Martin K,Steinberg TH,Cooley LA,Gee KR,Beechem JM,Patton WF||Proteomics (3:1244)||2003|