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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. Secondary antibodies are used throughout various types of assays, including ELISA or Western Blot, Immunohistochemistry, Flow Cytometry. The secondary antibody type is selected according to the class of the primary antibody (e.g., IgG or IgM), the source host, and the kind of label which is preferred. Most primary antibodies are of the IgG class and are produced in a common set of host species that includes rabbit, mouse, goat or chicken. Therefore, anti-mouse IgG, anti-rabbit IgG, anti-goat IgG or anti-chicken polyclonal antibodies are often used. Choosing a secondary antibody is straightforward: select a secondary antibody that recognizes the host species used to produce the primary antibody of interest, i.e. choose an anti-mouse secondary antibody to detect a mouse monoclonal primary antibody. However, identifying the optimal secondary antibody requires knowledge of the detection assay. For example: Western blot and ELISA can be performed using colorimetric, chemiluminescent, and fluorescence reporter systems, while immunofluorescence and flow cytometry are generally limited to fluorescent reporter labels. In all of these instances, a conjugated secondary antibody is required.
The following variables should be considered in the approximate order of importance:
Secondary Antibody Selection
Polyclonal antibodies are sufficient for most needs.
Monoclonal antibodies are more difficult to produce but are highly specific and yield more consistent results over time.
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
Attention should be paid to the host species that was used to generate the primary antibody. Researchers should select a secondary antibody specific for detection of the primary antibody species. For example, when using a polyclonal antibody produced by rabbit you will select an anti-rabbit secondary antibody that was raised in an alternate host species such as goat or donkey. Rockland offers a wide range of secondary options for almost every possible experimental protocol.
Common Host-Specific Secondary Antibodies from Rockland
For commonly used techniques such as Western blot and ELISA, an enzyme conjugated secondary is most likely the best choice. Good examples are Peroxidase or Alkaline phosphatase. (*Enzymes require a substrate for generation of signal output). In the case of immunoassays such as immunofluorescence microscopy or flow cytometry (also called FACS) it is more typical to use a secondary antibody conjugated to a fluorochrome (i.e. FITC, DyLight™ or Cy™ dye). For immunoprecipitation experiments a special product that does not detect the precipitating antibody is essential for best results. Rockland’s TrueBlot® products are useful for the accurate detection of secondary antibodies used for immunoprecipitation followed by western blot. TrueBlot® products help eliminate heavy/light chain blotting and contamination from immunoprecipitation that can obscure target detection. 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.
Secondary Antibody by ApplicationsSecondary antibodies ELISA Secondary antibodies Western Blot analysis Secondary antibodies Immunostaining Secondary antibodies Immunohistochemistry Secondary antibodies Immunocytochemistry Secondary antibodies Flow Cytometry
Polyclonal antibodies generated in rabbit, goat, donkey, or chicken and are usually IgG isotype and thus the secondary antibody should then be an anti-IgG H&L (i.e. both heavy and light chain specific) antibody. IgM is also used, but at less frequency. In certain experiments a pre adsorbed secondary antibody is needed (See Pre-Absorbed Antibodies below). Monoclonal primary antibodies are commonly raised in mouse, rat and Armenian hamster, although rabbit and human derived monoclonal antibodies are also popular. The main antibody classes are designated IgA (α), IgD (δ), IgE (ε), IgG (γ) and IgM (μ).
Mouse immunoglobulin classes and subclasses:- Classes: IgG, IgM- Subclasses:IgG1, IgG2a, IgG2b, IgG3; Types: κ light chain, λ light chainRat immunoglobulin classes and subclasses:- Classes: IgG, IgM- Subclasses IgG1, IgG2a, IgG2b , IgG2c; Types: κ light chain, λ light chainHuman immunoglobulin classes and subclasses- Classes: IgG, IgM, IgA, IgD- Subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2; Types κ light chain, λ light chain
F(ab')2 fragment 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. Rockland offers fragmentation as a custom service.
F(ab')2 fragment antibodies eliminate non-specific binding between the Fc portions of antibodies and the Fc receptors on cells. When 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 binding to Fc receptors present on many cells. F(ab')2 fragment conjugated secondary antibodies are ideal for Flow Cytometry, Immunohistochemistry and Immunofluorescence.
Schematic of F(ab’)2 antibody fragment. Heavy and light chain are indicated by the H and L, disulfide bonds are yellow
F(ab')2 Anti-GOAT IgG [H&L] (RABBIT) Antibody Alkaline Phosphatase Conjugated
These reagents are prepared from stocks of our highest affinity antibodies by special papain digestion and subsequent purification to yield the approximately 50 kDa monovalent Fab antibody portion of the molecule. Fab antibody reagents are then coupled to reporter molecules. These reagents lack the F(c) portion of the molecule and will not interact with either in-vitro or cellular F(c) binding mechanisms. The relative low molecular weight of these reagents maximizes permeability for in-situ studies.Fab fragment antibodies are useful in experiments where two primary antibodies from the same host species are used, such as two IgG-type mouse monoclonal antibodies, as an example, Cell Signaling or Phospho Specific Antibodies. A conjugated Fab fragment anti-Mouse IgG is applied which binds to the surface of the mouse IgG antibody molecule completely masking all sites for anti-Mouse IgG binding. In addition, the Fab fragment antibody, because of its monovalent properties, is not able to bind any additional Mouse IgG antibody if it were to be reintroduced into the system. At this point the second primary antibody, also Mouse IgG, is applied and subsequently detected with a second conjugated anti-Mouse IgG. Again, the masking properties of the conjugated Fab fragment antibody used to bind the first primary antibody shield the first Mouse IgG from detection by the second anti-Mouse antibody.Please inquire for other reporter molecule conjugates, such as, DyLight and IRDye or for Fab fragments of any other antibody listed in our catalog. Note: Anti-IgG F(ab')2 antibodies will recognize the Fab and F(ab')2 portions of the IgG molecule equally well.
Secondary antibodies can be general recognizing whole IgG and any fragments thereof, or can be specific recognizing only a single host species of primary antibody protein. For this reason Rockland has developed affinity purified and highly cross adsorbed secondary antibodies. Antibodies may be directed against either the whole molecule IgG, designated as "(H&L)" for heavy and light chains, or the only an antibody fragment that for example could include the light chain, F(c) portion of IgG, or the F(ab')2 portion of IgG. Pre-adsorbtion (also cross-adsorbtion) of the secondary antibody is used to eliminate reactivity to IgG from undesired host species or antibody fragments. The degree of cross reactivity is determined by ELISA and is typically less than 1% of the desired signal. 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 (i.e., 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.Many antibodies are offered as pre adsorbed against serum proteins from another species or are adsorbed against a mixture of serum proteins from several species (ie. designated "X to Ch,GP,Ham,Hs,Ms,Rb & Rt"). These highly cross adsorbed antibodies show extremely low levels of cross reactivity in multiple labeling experiments. The degree of cross reactivity is determined by ELISA and is typically less than 1% of the desired signal. See a list of pre-adsorbed antibodies offered by Rockland
Related products See all related substrate products from Rockland
• Peroxidase enzyme substrates for ELISA • Peroxidase enzyme substrates for WB and IHC • Blocking buffers • Chemiluminescent FemtoMax™ Super Sensitive HRP Substrate FEMTOMAX-110 • NPP (50X) ELISA Alkaline Phosphatase Substrate NPP-10
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