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Secondary Antibodies

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.

H+L stands for heavy and light chains. It means that the antibody will bind to epitopes on both the heavy chains (constant regions) and light chains (variable regions) of the target species immunoglobulin.

“Min x” followed by a list of species means that the antibody has minimal cross-reactivity against antibodies from those species. The antibody has been adsorbed against serum proteins from the listed species, to ensure that it does not cross-react with those species.

Highly cross-adsorbed antibodies are recommended when doing multiple staining with antibodies from different species (for example, co-staining with two different primary antibodies from mouse and rat hosts) or when staining tissues from species that are closely related to the antibody host (such as using mouse antibodies to stain rat tissue).

We recommend using highly cross-adsorbed secondary antibodies whenever you perform multiple staining with two primary antibodies from different hosts. It is particularly important to use highly cross-adsorbed antibodies when staining with antibodies from two closely related species, such as mouse and rat.

Using highly cross-adsorbed secondary antibodies when staining tissues from a species with primary antibodies from a closely related species (for example, staining rat tissue with mouse primary antibody), will prevent background from cross-reactivity of the secondary with endogenous immunoglobulin in the tissue. This is important when staining immune tissues, but background from endogenous immunoglobulin is common in other tissues around blood vessels, where immunoglobulin also is abundant.

As a rule, we recommend using secondary antibodies from the same host for multiplex staining (for example, combining Goat Anti-Rabbit with Goat Anti-Mouse, or Donkey Anti-Rabbit with Donkey Anti-Mouse). If we don’t currently offer the particular secondary antibody conjugate you wish to use, be sure to contact us, we may be able to perform a custom conjugation for you.

In our experience, cross-reactivity between donkey and goat host secondary antibodies is low, but this is not guaranteed to be the case for every lot of antibody. If you decide to combine secondary antibodies from different hosts, we recommend performing the appropriate controls to make sure the two secondary antibodies are not cross-reacting with each other.

No, antibodies designated as anti-IgG (H+L) with no subtype specified will cross react with all sub-types of IgG, as well as other isotypes like IgM, because they react with epitopes on the both the heavy (constant) and light (variable) regions of the antibody.

Secondary antibodies that react with IgG (H+L) will react with epitopes on both heavy and light chains, so they will react with other isotypes of primary antibody, such as IgM, or different subtypes of IgG (IgG2, IgG2a, etc.). Secondary antibodies that specify a specific isotype for their reactivity (such as Goat Anti-Mouse IgG2a, Isotype Specific) are cross-adsorbed against other isotypes for specific binding.

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.

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.

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.

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