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Secondary antibodies bind to the primary antibody to assist in detection, sorting and purification of target antigens. To enable detection, the secondary antibody must have specificity for the antibody species and isotype of the primary antibody being used and generally is conjugated. Primary antibodies are typically produced in mouse, rabbit, goat or chicken host species; therefore, anti-mouse IgG, anti-rabbit IgG, anti-goat IgG or anti-chicken polyclonal antibodies are used. Identifying the optimal secondary antibody is normally done based on the nature of the primary antibody and the detection assay. Secondary antibodies are used throughout various types of assays, including ELISA or Western Blot, the secondary antibody type is selected according to the source of the primary antibody, the class of the primary antibody (e.g., IgG or IgM), and the kind of label which is preferred.
The Mouse TrueBlot® Western Blot Kit contains the critical supporting reagents, buffers, and substrates for GFP immunoprecipitation and Western blotting of samples using TrueBlot second step immunoblotting reagents in conjunction with your own primary IP antibody and primary (Mouse IgG) Western blotting antibody. TrueBlot technology enables unhindered detection of protein bands of interest which would otherwise be obscured by the presence of reduced and denatured heavy and light chain immunoglobulin in the blot (as detected by the conventional immunoblotting HRP anti-mouse IgG reagent).
Horse Radish Peroxidase (HRP) Conjugated Secondary Antibody is the most popular conjugated antibody. It can be used in Western Blot Analysis (immunoblotting), ELISA and Immunohistochemistry. Beside the HRP conjugated secondary antibodies, Rockland further produces secondary antibodies that are FITC conjugated, as well as conjugates to Rhodamine, Texas Red and Phycoerythrin (PE) and dyes for multiplex platforms like the LI COR Odyssey Imaging System or Molecular Devices multi-mode readers. These dyes consist of Atto Dye, DyLight™ Dye, IRDye® 700 and IRDye® 800.
Secondary antibodies can be specific (e.g., recognize only one host species of primary antibody protein) or can be general (e.g., recognize whole IgG and any fragments thereof). For this reason Rockland has developed highly cross adsorbed secondary antibodies. The secondary antibody is cross adsorbed against serum antibody protein from another species or is adsorbed against a mixture of serum antibody protein from several species (e.g. Pre-adsorbed). These highly cross adsorbed antibodies show low levels of cross reactivity in multiple labeling experiments. Cross reactivity of Pre-adsorbed secondary antibodies is determined by ELISA or Western Blot Detection and is typically less than 1% or of the desired signal.
F(ab')2 fragment biotinylated secondary antibodies are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving intact the hinge region. F(ab')2 fragments have two antigen-binding F(ab) portions linked together by disulfide bonds, and therefore are divalent with a molecular weight of about 110 kDa. F(ab')2 fragment biotinylated antibodies eliminate non-specific binding between Fc portions of antibodies and Fc receptors on cells. If working with tissues or cells that have Fc receptors (spleen, blood, hematopoietic cells, leukocytes, etc.), choose an F(ab')2 fragment to eliminate non-specific anti biotin antibody binding through Fc receptors present on cells. F(ab')2 fragment conjugated secondary antibodies are ideal for Flow Cytometry, Immunohistochemistry and Immunofluorescence.
Anti-HUMAN IgG (H&L) (GOAT) Antibody(Min X Bv Ch Gt GP Ham Hs Ms Rb Rt &Sh Serum Proteins)
Anti-MOUSE IgG (H&L) (GOAT) Antibody
Anti-MOUSE IgG (H&L) (GOAT) Antibody(Min X Bv Ch Gt GP Ham Hs Hu Rb Rt &Sh Serum Proteins)
Anti-MONKEY IgG (gamma chain) (GOAT) Antibody
Anti-RABBIT IgG (H&L) (GOAT) Antibody
Anti-RABBIT IgG (H&L) (GOAT) Antibody(Min X Bv Ch Gt GP Ham Hs Hu Ms Rt & Sh Serum Proteins)
Anti-SHEEP IgG (H&L) (RABBIT) Antibody
F(ab')2 Anti-HUMAN IgM Fc5µ (GOAT) Antibody
Rockland offers a number of TrueBlot® products to assist in the accurate detection and isolation of secondary antibodies utilized in a variety of assays. TrueBlot® products help eliminate heavy/light chain blotting and contamination from immunoprecipitation that can derail many projects. TrueBlot® products offer increased sensitivity, less background noise, and enhanced accuracy for numerous applications. TrueBlot® reagents are available in several options, including individual IP Beads and complete IP/Western Blot kits from goat, mouse, rabbit or sheep.
Enzymatic Secondary Antibody Conjugates - Reporter enzymes are used extensively in molecular biology, allowing visualization or detection of immune complexes. Horseradish Peroxidase (HRP) is a widely used reporter enzyme, and depending on the substrate it can yield a chromogenic, or luminescent product. Alkaline phosphatase is also used, most typically as the reporter in chromogenic western blot assay format.
Fluorescent Secondary Antibody Conjugates - Rockland conjugates a broad group of secondary antibodies to many of the classic and next generation of fluorescent markers including fluorescein, Texas Red, and Phycoerythrin. All of the conjugates are ideal for various immunofluorescence based assays including fluorescent western blotting, immunofluorescence microscopy, FLISA, and more. Rockland also produces many next generation fluorochrome dyes designed for detection of primary antibodies in multiplex, multi-color analysis. Next generation fluorochrome conjugates (Atto-tec dyes, DyLight™ dyes) offer superior absorption (high extinction coefficient), high fluorescence quantum yield, and superior high photostability.
IRDye® Fluorescent Dye Conjugates - IRDye® infrared conjugated secondary antibodies (i.e.IRDye800, IRDye800CW, IRDye700 Antibody Conjugates) are often cross-adsorbed for multiplex detection and provide high specificity for assays such as fluorescence microscopy, flow cytometry, western blotting, ELISA, multiplex assays and other array platforms. These antibody conjugates are optimized for high sensitivity and excellent signal-to-noise performance on LI-COR Odyssey® imaging systems and other detection platforms.
Secondary, or indirect, immunofluorescence uses two antibodies; a primary antibody which recognizes the target biomolecule and binds to it and a secondary antibody conjugated to a fluorescent dye, which recognizes and binds to the primary antibody and indirectly localizes the target for detection by the microscope. While this protocol is more complex than the direct method, it is more flexible with regard to experimental design, results in greater signal detection through amplification and is easier in that secondary antibody conjugates are commercially available. Visit our Immunofluorescence Microscopy page for more information.
Rockland's Immunohistochemistry Studies (IHC) provide confidential high-quality target localization data through its extensive services. Researchers are presented with the option of outsourcing their entire IHC studies, bypassing the difficulties of sourcing and characterizing the appropriate target-specific antibodies or difficult-to-find validated tissues and the complex immunostain interpretation thereof. Commonly requested standardized IHC services are offered as Service Packets; however, Rockland’s custom design of your required study plan will address your particular biological questions. All studies are conducted under R&D conditions, are confidential and the results are proprietary to the customer. Visit our Immunohistochemistry Studies page for more information.
Oftentimes secondary antibodies recognize only one host species of primary antibody or alternatively recognize the entire IgG and any fragments thereof. Choosing the appropriate secondary probe or sufficiently designing the experimental system can overcome this disparity. When choosing a new antibody, keep the following considerations in mind:
Protect your experiments with Rockland secondary antibodies. Compromise elsewhere. All Secondary Antibodies are backed by our RockSolid Antibody Guarantee.
Rockland Immunochemicals Inc.Gilbertsville, PA 19525E-mail: email@example.comPhone: 800.656.7625