Immunofluorescence analysis of Goat anti-Mouse IgG1 Secondary Antibody, Alexa Fluor 568 conjugate (Product # A21124) was performed using HeLa cells stained with alpha Tubulin (236-10501) Mouse Monoclonal Antibody (Product # 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 mouse primary antibody (1:250 dilution) for 3 hours at room temperature. Goat anti-Mouse IgG1 Secondary Antibody, Alexa Fluor 568 conjugate was used at concentration of 4µg/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: green). Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (Product # S36938). F-actin was stained with Alexa Fluor® 488 Phalloidin (Product # A12379, 1:300) (Panel c: red). 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||IgG gamma 1|
|Conjugate||Alexa Fluor® 568|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Cross Adsorption||Against mouse IgM, mouse IgA, pooled human sera, purified human paraproteins and mouse isotypes IgG2a, IgG2b and IgG3 prior to conjugation|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:500|
|Immunocytochemistry (ICC)||4 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.
|Immunocytochemistry (ICC)||See 1 publications below|
|Immunohistochemistry (Paraffin) (IHC (P))||See 1 publications below|
|Immunohistochemistry (IHC)||See 2 publications below|
|Immunohistochemistry (Frozen) (IHC (F))||See 1 publications below|
|Miscellaneous PubMed (MISC)||See 9 publications below|
To minimize cross-reactivity, these goat anti-mouse IgG1 whole secondary antibodies have been affinity purified and cross-adsorbed against mouse IgM, mouse IgA, pooled human sera, purified human paraproteins, and mouse isotypes IgG2a, IgG2b, and IgG3 prior to conjugation. 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 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||
Establishment and Biological Characterization of a Panel of Glioblastoma Multiforme (GBM) and GBM Variant Oncosphere Cell Lines.
A-21124 was used in immunocytochemistry to characterize a panel of glioblastoma multiforme (GBM) and GBM variant oncosphere cell lines by establishment and biological characterization
|Binder ZA,Wilson KM,Salmasi V,Orr BA,Eberhart CG,Siu IM,Lim M,Weingart JD,Quinones-Hinojosa A,Bettegowda C,Kassam AB,Olivi A,Brem H,Riggins GJ,Gallia GL||PloS one (11:null)||2016|
|Not Applicable||Not Cited||
FOXE3 mutations predispose to thoracic aortic aneurysms and dissections.
A-21124 was used in immunohistochemistry - paraffin section to investigate the predisposition of thoracic aortic aneurysms and dissections by FOXE3 mutations
|Kuang SQ,Medina-Martinez O,Guo DC,Gong L,Regalado ES,Reynolds CL,Boileau C,Jondeau G,Prakash SK,Kwartler CS,Zhu LY,Peters AM,Duan XY,Bamshad MJ,Shendure J,Nickerson DA,Santos-Cortez RL,Dong X,Leal SM,Majesky MW,Swindell EC,Jamrich M,Milewicz DM||The Journal of clinical investigation (126:948)||2016|
Quantitative Differences in Nuclear ß-catenin and TCF Pattern Embryonic Cells in C. elegans.
A-21124 was used in immunohistochemistry to characterize quantitative differences in C. elegans regarding nuclear beta-catenin and TCF pattern embryonic cells
|Zacharias AL,Walton T,Preston E,Murray JI||PLoS genetics (11:null)||2015|
Pathways and genes differentially expressed in the motor cortex of patients with sporadic amyotrophic lateral sclerosis.
A-21124 was used in immunohistochemistry to discuss screening the human motor cortex for therapeutic targets of amyotrophic lateral sclerosis
|Lederer CW,Torrisi A,Pantelidou M,Santama N,Cavallaro S||BMC genomics (8:null)||2007|
|Not Applicable||Not Cited||
Lamin A/C mutants disturb sumo1 localization and sumoylation in vitro and in vivo.
A-21124 was used in immunohistochemistry - frozen section to suggest that mutant lamin A/C alters the dynamics of sumo1 which contributes to laminopathies
|Boudreau É,Labib S,Bertrand AT,Decostre V,Bolongo PM,Sylvius N,Bonne G,Tesson F||PloS one (7:null)||2012|
|Not Applicable||Not Cited||Mucin5B expression by lung alveolar macrophages is increased in long-term smokers.||Sepper R,Prikk K,Metsis M,Sergejeva S,Pugatsjova N,Bragina O,Marran S,Fehniger TE||Journal of leukocyte biology (92:319)||2012|
|Not Applicable||Not Cited||Basolateral Cl- uptake mechanisms in Xenopus laevis lung epithelium.||Berger J,Hardt M,Clauss WG,Fronius M||American journal of physiology. Regulatory, integrative and comparative physiology (299:R92)||2010|
|Not Applicable||Not Cited||Retention of Pax3 expression in satellite cells of muscle spindles.||Kirkpatrick LJ,Yablonka-Reuveni Z,Rosser BW||The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society (58:317)||2010|
|Not Applicable||Not Cited||Dominant role of CD47-thrombospondin-1 interactions in myeloma-induced fusion of human dendritic cells: implications for bone disease.||Kukreja A,Radfar S,Sun BH,Insogna K,Dhodapkar MV||Blood (114:3413)||2009|
|Not Applicable||Not Cited||Improved prediction of prostate cancer recurrence through systems pathology.||Cordon-Cardo C,Kotsianti A,Verbel DA,Teverovskiy M,Capodieci P,Hamann S,Jeffers Y,Clayton M,Elkhettabi F,Khan FM,Sapir M,Bayer-Zubek V,Vengrenyuk Y,Fogarsi S,Saidi O,Reuter VE,Scher HI,Kattan MW,Bianco FJ,Wheeler TM,Ayala GE,Scardino PT,Donovan MJ||The Journal of clinical investigation (117:1876)||2007|
|Not Applicable||Not Cited||CD1d degradation in Chlamydia trachomatis-infected epithelial cells is the result of both cellular and chlamydial proteasomal activity.||Kawana K,Quayle AJ,Ficarra M,Ibana JA,Shen L,Kawana Y,Yang H,Marrero L,Yavagal S,Greene SJ,Zhang YX,Pyles RB,Blumberg RS,Schust DJ||The Journal of biological chemistry (282:7368)||2007|
|Not Applicable||Not Cited||Phosphorylation controls CLIMP-63-mediated anchoring of the endoplasmic reticulum to microtubules.||Vedrenne C,Klopfenstein DR,Hauri HP||Molecular biology of the cell (16:1928)||2005|
|Not Applicable||Not Cited||Different MHC class I heavy chains compete with each other for folding independently of beta 2-microglobulin and peptide.||Tourdot S,Nejmeddine M,Powis SJ,Gould KG||Journal of immunology (Baltimore, Md. : 1950) (174:925)||2005|
|Not Applicable||Not Cited||Mouse model carrying H222P-Lmna mutation develops muscular dystrophy and dilated cardiomyopathy similar to human striated muscle laminopathies.||Arimura T,Helbling-Leclerc A,Massart C,Varnous S,Niel F,Lacène E,Fromes Y,Toussaint M,Mura AM,Keller DI,Amthor H,Isnard R,Malissen M,Schwartz K,Bonne G||Human molecular genetics (14:155)||2005|