Secondary Antibodies

Rockland offers a wide range of secondary options for almost every possible experimental protocol. Their secondary antibodies are sourced from a variety of hosts in their onsite vivarium.

Secondary antibodies are used with various types of assays, including ELISA, Western blot (WB), immunohistochemistry (IHC), and flow cytometry (FC). 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 such as horseradish peroxidase (HRP). 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.

Types of secondary antibodies include:

• Conjugated Antibodies
• Fab Fragmented Antibodies
• Pre-adsorbed Antibodies

Applications

• ELISA
• Dot Blot
• Western Blot
• SDS-PAGE
• Purification
• IP
• Cellular Assay
• IHC
• IF
• FC
• Biochemical Assay

BROWSE ALL SECONDARY ANTIBODIES

Conjugated Antibodies

Conjugated antibodies are high-performance monoclonal or polyclonal antibodies coupled to colorimetric, chemiluminescent or fluorescent labels for use as reporter molecules in immunoassays. Rockland’s conjugated antibodies are designed to be used in a variety of applications, such as ELISA, FLISA, Western blot, immunofluorescence, immunohistochemistry, flow cytometry, and others while working across multiple imaging platforms.

Through continued scientific collaborations with academic institutions, Rockland has developed hundreds of conjugated primary antibody and secondary antibody reagents, including AKT and GFP antibodies in response to market demands.

Rockland produces conjugated primary antibodies and conjugated secondary antibodies to reporter molecules like enzymes, fluorescent dyes, infrared dyes, chemiluminescent reporters, and haptens for use in commonly used immunoassays.

Fab Fragment Antibodies

Fab fragment antibodies are generated by papain digestion and subsequent purification to yield the approximately 50 kDa monovalent Fab antibody portion of the molecule. These fragments lack the Fc portion of the molecule and will not interact with either in vitro or cellular Fc binding mechanisms. The relatively 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. 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, because of the Fab fragment antibody’s monovalent properties, it is not able to bind any additional mouse IgG antibody if it were 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. 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 mouse antibody.

F(ab’)2 Fragment Antibodies

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 palent with a molecular weight of about 110 kDa.

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. F(ab’)2 fragment conjugated secondary antibodies are ideal for flow cytometry, immunohistochemistry, and immunofluorescence.

Pre-adsorbed Antibodies

Pre-adsorption (also cross-adsorption) 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 cross-adsorbed antibodies show low levels of cross-reactivity in multiple labeling or multiplex experiments. Cross-reactivity of pre-adsorbed secondary antibodies is determined by ELISA or Western blot detection.

Below are our most popular pre-adsorbed secondary antibodies by class and subclass.

Mouse immunoglobulin classes and subclasses:
Classes: IgG, IgM
Subclasses: IgG1, IgG2a, IgG2b, IgG3; Types: κ light chain, λ light chain

Rat immunoglobulin classes and subclasses:
Classes: IgG, IgM
Subclasses: IgG1, IgG2a, IgG2b , IgG2c; Types: κ light chain, λ light chain

Human immunoglobulin classes and subclasses:
Classes: IgG, IgM, IgA, IgD
Subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2; Types κ light chain, λ light chain

Pre-absorbed Antibodies for Multiplex Assays

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 secondary 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 allowing for the use of several primary antibodies for different host species in one assay..

Brand: Rockland Immunochemicals Inc
Categories: Antibodies, Media, Kits and Other Reagents, Antibodies