| Product Specific InformationTo minimize cross-reactivity, the goat anti-rabbit IgG whole antibodies have been highly 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. Further passages through additional columns result in 'highly cross-adsorbed' preparations of secondary antibody. The benefits of these extra steps are apparent in multiplexing/multicolor-staining experiments where there is potential cross-reactivity with other primary antibodies or in tissue/cell fluorescent staining experiments where there 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.Background/Target InformationWe 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. |
Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (A-11034) in IFImmunofluorescence analysis of Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor® 488 conjugate was performed using HeLa cells stained with alpha Tubulin Rabbit Polyclonal Antibody (Product # PA5-16891). 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 2 µg/ml Rabbit primary antibody for 3 hours at room temperature. Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor® 488 conjugate (Product # A-11034) was used at a concentration of 4 µg/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 Rhodamine Phalloidin (Product # R415, 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.Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (A-11034) in IFA 2.0 µm maize leaf section illustrating the immunolocalization of the enzyme ribulose bisphosphate carboxylase (rubisco) in the chloroplasts of the bundle sheath cells surrounding the vascular bundles. Maize is a C4 plant and, as a result, spatially segregates components of the photosynthetic process between the leaf mesophyll and the bundle sheath. Rubisco was localized using a rabbit anti-rubisco antibody and visualized using the highly cross-adsorbed Alexa Fluor® 488 goat anti-rabbit IgG antibody (Product # A-11034). The remaining fluorescence is due to the autofluorescence of chlorophyll, which appears red and is localized to the mesophyll plastids; lignin, which appears dull green and is localized to the xylem of the vascular bundle; and cutin, which appears bright green and is localized to the cuticle outside the epidermis. Image contributed by Todd Jones, DuPont.Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (A-11034) in IFHuman iPSC Staining Human iPSCs were cultured on glass slides under feeder-free conditions in StemPro® hESC Medium (Product # A1000701). Cells were fixed and permed with the Image-iT® Fixation/Permeabilization Kit (Product # R37602). Oct4 (green) expression was visualized using anti-Oct4 primary Ab and Alexa Fluor® 488 secondary Ab (Product # A-11034). Tubulin (red) expression was visualized using anti-tubulin primary Ab (Product # 32-2600) and Alexa Fluor® 594 secondary Ab (Product # A-11005). Nuclei (blue) were labeled with NucBlue™ Fixed Cell Stain (Product # R37606). Images were collected on the FLoid™ Cell Imaging Station (Product # 4471136). |
| 1.Frontiers in aging neuroscienceIncrease of TREM2 during Aging of an Alzheimer's Disease Mouse Model Is Paralleled by Microglial Activation and Amyloidosis."A11034 was used in immunohistochemistry to quantify soluble triggering receptor expressed on myeloid cells 2 and amyloid levels in Alzheimer's disease mouse model brains"Authors Brendel M,Kleinberger G,Probst F,Jaworska A,Overhoff F,Blume T,Albert NL,Carlsen J,Lindner S,Gildehaus FJ,Ozmen L,Suárez-Calvet M,Bartenstein P,Baumann K,Ewers M,Herms J,Haass C,Rominger A2.eLifeDirect measurement of TRPV4 and PIEZO1 activity reveals multiple mechanotransduction pathways in chondrocytes."A11034 was used in immunohistochemistry to discover there are separate, but overlapping, mechanoelectrical transduction pathways in chondrocytes"Authors Servin-Vences MR,Moroni M,Lewin GR,Poole K3.Scientific reportsG196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity."A11034 was used in immunocytochemistry to design a new epitope tag family specifically recognized by the monoclonal antibody G196"Authors Tatsumi K,Sakashita G,Nariai Y,Okazaki K,Kato H,Obayashi E,Yoshida H,Sugiyama K,Park SY,Sekine J,Urano T4.Journal of cell scienceIncreased ROS production in non-polarized mammary epithelial cells induces monocyte infiltration in 3D culture."A-11034 was used in immunocytochemistry to propose that increased ROS production in mammary epithelial cell disrupts cell polarity and promotes monocyte infiltration"Authors Li L,Chen J,Xiong G,St Clair DK,Xu W,Xu R5.Scientific reportsImmunoscreening of Plasmodium falciparum proteins expressed in a wheat germ cell-free system reveals a novel malaria vaccine candidate."A11034 was used in immunocytochemistry to establish LSA3 as a novel blood-stage vaccine candidate against Plasmodium falciparum"Authors Morita M,Takashima E,Ito D,Miura K,Thongkukiatkul A,Diouf A,Fairhurst RM,Diakite M,Long CA,Torii M,Tsuboi T |
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