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Unless specified as isotype-specific, anti-IgG secondary antibodies are raised against whole immunoglobulin, so they are expected to cross-react widely with isotypes other than IgG.

No, you don’t always need to use serum from the same species as the secondary antibody for blocking. BSA, fish gelatin, goat serum, or non-fat milk (for western) can be used with secondary antibodies from most host species. However, it is important to avoid using blocking serum from the same host species as your primary antibody that is detected by a secondary antibody against the same species (immunoglobulins in the blocking serum will compete for the secondary binding). For example, avoid using goat serum for blocking if you are using a goat primary antibody with anti-goat secondary antibody.

Our TrueBlack® IF Background Suppressor System and TrueBlack® WB Blocking Buffer Kit contain proteins from non-mammalian sources, and should not interact with secondary antibodies from mammalian or chicken host. Our BSA also is immunoglobulin-free and should not interfere with secondary antibodies.

F(ab) fragment-specific antibodies react with the light chains and not the heavy chains of immunoglobulins from the target species. They can be used to detect antibody fragments, or to avoid cross-reactivity with immunoglobulin heavy chains.

F(ab’)2 is a fragment of IgG that is prepared by pepsin digestion of IgG. F(ab’)2 fragment is the disulfide-linked heterodimer of the two light chain dimers, so it retains bivalent epitope binding like whole IgG, but because it lacks the heavy chains, it is smaller in size (~110 kDa compare to 150 kDa for whole IgG).

F(ab’) is a monovalent fragment consisting of a single light chain homodimer, which is obtained by pepsin digestion of IgG, followed by reduction of the light chain disulfide bond.

F(ab’)2 and F(ab’) fragments do not bind to immunoglobulin receptors on cells, which can be useful for achieving specific staining of the primary antibody target. The fragments also will not bind Protein A or Protein G.

IgY is the avian counterpart to mammalian IgG, or the type of antibody produced in birds. Goat Anti-Chicken IgY (H+L) is a secondary antibody raised in a goat host that reacts with both heavy and light chains of chicken antibodies.

Isotype-specific antibodies (for example, Goat Anti-Mouse IgG1, Goat Anti-Mouse IgG2a, or Goat Anti-Mouse IgG2b) can be used to specifically detect primary antibodies from the listed sub-class. This can be useful for multiplex detection using multiple mouse monoclonal antibodies that are different isotypes.

Anti-mouse subtype-specific secondary antibodies also can be useful for avoiding background when staining mouse tissues with mouse monoclonal antibodies, because IgG is present at relatively low levels in most mouse tissues.

Isotype-specific antibodies also can be used for identifying the isotype of a monoclonal antibody, or for staining B-cell subsets.