{"id":2539,"date":"2018-08-13T04:43:53","date_gmt":"2018-08-13T08:43:53","guid":{"rendered":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/?p=2539"},"modified":"2021-04-09T05:14:26","modified_gmt":"2021-04-09T09:14:26","slug":"species-on-species-experiments-solutions","status":"publish","type":"post","link":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/technical-tips\/species-on-species-experiments-solutions\/","title":{"rendered":"Solutions for Species on Species Experiments"},"content":{"rendered":"\n\n\n\t
\n\n\t\t<\/i><\/span> Download PDF<\/a>\n\n\t<\/div>\n\n\n\n\n\n

When using primary antibodies derived from the same species as the sample material (species on species)\u00a0 non-specific background staining can occur. Mouse on mouse staining (M.O.M. staining) is a common example of the problem. This article explains how you can prevent background signal in Species on Species experiments.<\/h2>\n

The species on species problem<\/b><\/h4>\n

The species on species problem occurs when using a secondary antibody to visualize a primary antibody on a sample derived from the same species as the primary antibody.<\/span><\/p>\n

Endogenous immunoglobulins presented by the tissue are recognized by the secondary antibody, which is unable to distinguish between primary antibodies and endogenous immunoglobulins of the same species. The resulting non-specific staining can obscure signal from the primary antibody or reveal a misleading staining pattern. <\/span><\/p>\n

JIR offers tools to help you avoid background when conducting species on species experiments.<\/span><\/p>\n

Solutions for species on species labeling<\/b><\/h4>\n

Monovalent Fab fragments can be used to \u201cmask\u201d or block the endogenous Ig epitopes, making them unavailable for detection by the labeled secondary antibody. Fab fragments have a single antigen binding site, so they will not capture the primary antibody used in a subsequent step. In contrast, whole molecule IgG and F(ab\u2019)2<\/sub> fragments are divalent (they have two binding sites) and are ineffective for blocking endogenous Ig. After binding the endogenous Ig with one arm, a divalent antibody may have an open binding site which could capture the primary antibody introduced in a later step.<\/span><\/p>\n

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Figure 1: The digestion of IgG (approximate MW = 160 kDa) yields a number of different fragments. Papain digestion generates two monovalent Fab fragments and an Fc fragment, each about 50 kDa. Pepsin digestion degrades the Fc domain, leaving a divalent F(ab\u2032)2<\/sub> fragment with approximate MW of 110 kDa.<\/em><\/p><\/div>\n

Choose a Fab fragment raised in the same host as <\/span>t<\/span>he labeled secondary antibody, e.g. Fab fragment Goat Anti-Mouse IgG (H+L) to block binding of labeled Goat Anti-Mouse IgG. Maintaining the same host species ensures non-reactivity between the Fab fragment and labeled secondary antibody. In addition, a Fab fragment raised in the same species will recognize, and therefore block, the same epitopes that the labeled secondary would recognize. A different host animal may recognize different epitopes on the endogenous Ig, and blocking could be incomplete.<\/span><\/p>\n

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Browse Fab Fragment Secondary Antibodies<\/a><\/p>\n

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To block endogenous immunoglobulins on cells or tissue sections<\/strong><\/h4>\n