Flow cytometry analysis of Goat anti-Mouse IgG1 Secondary Antibody, Alexa Fluor 647 conjugate (A21240) was performed using K-562 cells stained with alpha Tubulin Mouse Monoclonal Antibody (A11126). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with alpha Tubulin antibody or with mouse isotype control at 3-5 ug/million cells in 2.5% BSA and incubated for 2 hours at room temperature. The cells were then labeled with Goat anti-Mouse IgG1 Secondary Antibody, Alexa Fluor 647 conjugate (A21240) at a dilution of 1:500 for 1 hour at room temperature. A representative 10,000 cells were acquired and analyzed for each sample using the Attune® Acoustic Focusing Cytometer. Panel a represents cells stained with the secondary antibody alone (green histogram), isotype control (pink histogram) and alpha Tubulin Monoclonal Antibody (red histogram). Median fluorescence intensity from the three samples is compared in panel b.
|Tested species reactivity||Mouse|
|Published species reactivity||Not Applicable|
|Host / Isotype||Goat / IgG|
|Immunogen||IgG gamma 1|
|Conjugate||Alexa Fluor® 647|
|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)||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.
|Miscellaneous PubMed (MISC)||See 8 publications below|
To minimize cross-reactivity, these goat anti-mouse IgG1 whole secondary antibodies have been affinity purified and cross-adsorbed against mouse 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 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||Monocyte subset accumulation in the human heart following acute myocardial infarction and the role of the spleen as monocyte reservoir.||van der Laan AM,Ter Horst EN,Delewi R,Begieneman MP,Krijnen PA,Hirsch A,Lavaei M,Nahrendorf M,Horrevoets AJ,Niessen HW,Piek JJ||European heart journal (35:376)||2014|
|Not Applicable||Not Cited||The amino terminus of herpes simplex virus 1 glycoprotein K is required for virion entry via the paired immunoglobulin-like type-2 receptor alpha.||Chowdhury S,Chouljenko VN,Naderi M,Kousoulas KG||Journal of virology (87:3305)||2013|
|Not Applicable||Not Cited||Mutual regulation between SIAH2 and DYRK2 controls hypoxic and genotoxic signaling pathways.||Pérez M,García-Limones C,Zapico I,Marina A,Schmitz ML,Muñoz E,Calzado MA||Journal of molecular cell biology (4:316)||2012|
|Not Applicable||Not Cited||High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents.||Ma J,Guo L,Fiene SJ,Anson BD,Thomson JA,Kamp TJ,Kolaja KL,Swanson BJ,January CT||American journal of physiology. Heart and circulatory physiology (301:H2006)||2011|
|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||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||Inhibition of T cell activation by cyclic adenosine 5'-monophosphate requires lipid raft targeting of protein kinase A type I by the A-kinase anchoring protein ezrin.||Ruppelt A,Mosenden R,Grönholm M,Aandahl EM,Tobin D,Carlson CR,Abrahamsen H,Herberg FW,Carpén O,Taskén K||Journal of immunology (Baltimore, Md. : 1950) (179:5159)||2007|
|Not Applicable||Not Cited||Tamoxifen but not 4-hydroxytamoxifen initiates apoptosis in p53(-) normal human mammary epithelial cells by inducing mitochondrial depolarization.||Dietze EC,Caldwell LE,Grupin SL,Mancini M,Seewaldt VL||The Journal of biological chemistry (276:5384)||2001|