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Alexa Fluor® 488 (Cat. No. A11017) or fluorescein (Cat. No. F11021) conjugates of goat anti–mouse IgG antibody F(ab"e;)2 fragment were used to detect HEp-2 cells probed with human anti-nuclear antibodies. Samples were continuously illuminated and images were collected every 5 sec with a cooled CCD camera. Normalized intensity data demonstrate the difference in photobleaching rates.
|Tested species reactivity||Mouse|
|Published species reactivity||Not Applicable|
|Host / Isotype||Goat / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 488|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Cross Adsorption||Against human IgG and serum|
|Antibody Form||F(ab')2 Fragment|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1-10 µg/mL|
|Immunocytochemistry (ICC)||1-10 µg/mL|
|Immunofluorescence (IF)||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 22 publications below|
To minimize cross-reactivity, these goat anti-mouse IgG divalent F(ab')2 fragment antibodies have been cross-adsorbed against 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 488 dye is a bright, green-fluorescent dye with excitation ideally suited to the 488 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 488 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 488 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 reproducible technique for specific labeling of antigens using preformed fluorescent molecular IgG-F(ab')2 complexes from primary antibodies of the same species.||Owen GR,Häkkinen L,Wu C,Larjava H||Microscopy research and technique (73:623)||2010|
|Not Applicable||Not Cited||Super-resolution imaging with small organic fluorophores.||Heilemann M,van de Linde S,Mukherjee A,Sauer M||Angewandte Chemie (International ed. in English) (48:6903)||2009|
|Not Applicable||Not Cited||Heterogeneity in macrophage phagocytosis of Staphylococcus aureus strains: high-throughput scanning cytometry-based analysis.||DeLoid GM,Sulahian TH,Imrich A,Kobzik L||PloS one (4:null)||2009|
|Not Applicable||Not Cited||Mechanisms of quantum dot nanoparticle cellular uptake.||Zhang LW,Monteiro-Riviere NA||Toxicological sciences : an official journal of the Society of Toxicology (110:138)||2009|
|Not Applicable||Not Cited||Biosensor detection systems: engineering stable, high-affinity bioreceptors by yeast surface display.||Richman SA,Kranz DM,Stone JD||Methods in molecular biology (Clifton, N.J.) (504:323)||2009|
|Not Applicable||Not Cited||Efficient delivery of antibody into living cells using a novel HVJ envelope vector system.||Kondo Y,Fushikida K,Fujieda T,Sakai K,Miyata K,Kato F,Kato M||Journal of immunological methods (332:10)||2008|
|Not Applicable||Not Cited||Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4.||Ménager MM,Ménasché G,Romao M,Knapnougel P,Ho CH,Garfa M,Raposo G,Feldmann J,Fischer A,de Saint Basile G||Nature immunology (8:257)||2007|
|Not Applicable||Not Cited||A low molecular weight agonist signals by binding to the transmembrane domain of thyroid-stimulating hormone receptor (TSHR) and luteinizing hormone/chorionic gonadotropin receptor (LHCGR).||Jäschke H,Neumann S,Moore S,Thomas CJ,Colson AO,Costanzi S,Kleinau G,Jiang JK,Paschke R,Raaka BM,Krause G,Gershengorn MC||The Journal of biological chemistry (281:9841)||2006|
|Not Applicable||Not Cited||Revealing the topography of cellular membrane domains by combined atomic force microscopy/fluorescence imaging.||Frankel DJ,Pfeiffer JR,Surviladze Z,Johnson AE,Oliver JM,Wilson BS,Burns AR||Biophysical journal (90:2404)||2006|
|Not Applicable||Not Cited||LacSwitch II regulation of connexin43 cDNA expression enables gap-junction single-channel analysis.||Zhong G,Mantel PL,Jiang X,Jarry-Guichard T,Gros D,Labarrere C,Moreno AP||BioTechniques (34:1034)||2003|
|Not Applicable||Not Cited||Negative regulation of T cell activation by placental protein 14 is mediated by the tyrosine phosphatase receptor CD45.||Rachmilewitz J,Borovsky Z,Riely GJ,Miller R,Tykocinski ML||The Journal of biological chemistry (278:14059)||2003|
|Not Applicable||Not Cited||Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution.||Schenk S,Hintermann E,Bilban M,Koshikawa N,Hojilla C,Khokha R,Quaranta V||The Journal of cell biology (161:197)||2003|
|Not Applicable||Not Cited||A novel IkappaB protein, IkappaB-zeta, induced by proinflammatory stimuli, negatively regulates nuclear factor-kappaB in the nuclei.||Yamazaki S,Muta T,Takeshige K||The Journal of biological chemistry (276:27657)||2001|
|Not Applicable||Not Cited||Blockade of alpha 5 beta 1 integrins reverses the inhibitory effect of tenascin on chemotaxis of human monocytes and polymorphonuclear leukocytes through three-dimensional gels of extracellular matrix proteins.||Loike JD,Cao L,Budhu S,Hoffman S,Silverstein SC||Journal of immunology (Baltimore, Md. : 1950) (166:7534)||2001|
|Not Applicable||Not Cited||Endothelial cell surface F1-F0 ATP synthase is active in ATP synthesis and is inhibited by angiostatin.||Moser TL,Kenan DJ,Ashley TA,Roy JA,Goodman MD,Misra UK,Cheek DJ,Pizzo SV||Proceedings of the National Academy of Sciences of the United States of America (98:6656)||2001|
|Not Applicable||Not Cited||Identification of an amino acid residue in multidrug resistance protein 1 critical for conferring resistance to anthracyclines.||Zhang DW,Cole SP,Deeley RG||The Journal of biological chemistry (276:13231)||2001|
|Not Applicable||Not Cited||gC1q-R/p33, a member of a new class of multifunctional and multicompartmental cellular proteins, is involved in inflammation and infection.||Ghebrehiwet B,Lim BL,Kumar R,Feng X,Peerschke EI||Immunological reviews (180:65)||2001|
|Not Applicable||Not Cited||Molecular cloning and characterization of endosialin, a C-type lectin-like cell surface receptor of tumor endothelium.||Christian S,Ahorn H,Koehler A,Eisenhaber F,Rodi HP,Garin-Chesa P,Park JE,Rettig WJ,Lenter MC||The Journal of biological chemistry (276:7408)||2001|
|Not Applicable||Not Cited||Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control.||Zuber C,Spiro MJ,Guhl B,Spiro RG,Roth J||Molecular biology of the cell (11:4227)||2000|
|Not Applicable||Not Cited||Effects of incorporation of immunoglobulin G and complement component C1q on uptake and degradation of Alzheimer's disease amyloid fibrils by microglia.||Brazil MI,Chung H,Maxfield FR||The Journal of biological chemistry (275:16941)||2000|
|Not Applicable||Not Cited||Pathogenic mycobacteria disrupt the macrophage actin filament network.||Guérin I,de Chastellier C||Infection and immunity (68:2655)||2000|
|Not Applicable||Not Cited||AKAP350, a multiply spliced protein kinase A-anchoring protein associated with centrosomes.||Schmidt PH,Dransfield DT,Claudio JO,Hawley RG,Trotter KW,Milgram SL,Goldenring JR||The Journal of biological chemistry (274:3055)||1999|