Affinity-purified antibodies are isolated from antisera by immunoaffinity chromatography using antigens coupled to agarose beads. A proprietary, sequential elution process is used to dissociate antibodies from the antigen. Uncongugated affinity-purified antibodies are sterile filtered in phosphate buffer without stabilizers or preservatives. Conjugated affinity-purified antibodies are freeze dried in phosphate buffer with stabilizers and sodium azide as a preservative, with the exception of horseradish peroxidase conjugates, which do not contain a preservative.

Step 1.   Select either "Whole IgG ", "F(ab')₂ Fragments", or "Fab Fragments" of secondary antibodies.

Affinity-purified secondary antibodies are offered in three different forms: whole IgG, F(ab')₂ fragments, and Fab
fragments.

Whole IgG antibodies are isolated as intact molecules from antisera by affinity chromatography. They have an Fc portion and two antigen binding Fab portions joined together by disulfide bonds (Figure 1), and therefore are divalent. The average molecular weight is reported to be about 160 kD. The whole IgG form of antibodies is suitable for the majority of immunodetection procedures, and is the most cost effective.

F(ab')₂ fragments of antibodies are generated by pepsin digestion of whole IgG antibodies to remove a portion of the Fc region while leaving intact some of the hinge region.
F(ab')₂ fragments have two antigen-binding Fab portions linked together by disulfide bonds, and therefore are divalent (Figure 1). The average molecular weight is about 110,000 kD. They are used for specific applications, such as to avoid binding of antibodies to Fc receptors on cell surfaces or binding to Protein A or Protein G.

However, binding of primary antibodies to Fc receptors also may occur if they are whole IgG antibodies, creating background regardless of the form of the secondary antibody. To block whole IgG primary and whole IgG secondary antibodies from binding to Fc receptors, incubate cells in buffer containing 5% normal serum from the host species of the secondary antibody. To prevent capping, endocytosis, and regeneration of Fc receptors on living cells, incubate at 4°C in buffer containing 5% normal serum, with sodium azide added to inhibit metabolism. Since this procedure should be used with the majority of primary antibodies

Cautions:  Never block with normal serum or IgG from the host species of the primary antibody when using a labeled secondary antibody. If immunoglobulins in the normal serum bind to the specimen of interest, they will be recognized by the labeled secondary antibody, resulting in higher background.

Bovine serum albumin (BSA) and dry milk, both commonly used for blocking, may contain bovine IgG. With the exception of Bovine anti-Goat IgG, many secondary antibodies such as anti-bovine, anti-goat, and anti-sheep will react strongly with bovine IgG. Therefore, use of BSA or dry milk for blocking or diluting these antibodies may significantly increase background and/or reduce antibody titer. For blocking, use normal serum (5% v/v) from the host species of the secondary antibody.

Fab fragments of antibodies are generated by papain digestion of whole IgG antibodies to remove the entire Fc portion, including the hinge region (Figure 1). These antibodies are monovalent, containing only a single antigen binding site. The molecular weight of Fab fragments is about 50 kD. They can be used to block immunoglobulins on cells, tissues, or other surfaces.

In contrast, a divalent (whole IgG or F(ab')₂ fragment) antibody should not be used for blocking since it has two binding sites. After blocking, some of the binding sites may remain open to capture the primary antibody introduced in a subsequent step, and thus create higher background or result in coincidental labeling.

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The antibodies are listed alphabetically according to the host species of the antigen (i.e. the primary antibody). For example, if the primary antibody is made in mouse, go to the "Anti-Mouse" section.

Note: Both anti-Syrian and anti-Armenian hamster secondary antibodies are listed under "Anti-Hamster". It is important to know in which strain of hamster the primary antibody was produced in order to select the proper secondary antibody, since cross-reaction between the strains is not complete.

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Step 3.  Select the host species of the secondary antibody under the column labeled "Host".

The host species of the secondary antibody is in the beginning of the antibody description. Selection of the host species for a secondary antibody involves many considerations, including but not limited to: 1). Personal preference or experience. In our experience there appears to be no species specific difference in the quality of secondary antibodies. 2). Binding to Protein A and Protein G. Rabbit antibodies bind well to Protein A and G, but goat and donkey antibodies bind better to Protein G. 3). Availability of antibodies adsorbed against desired species. Antibodies from some host species may not be adsorbed against cross-reacting species of interest. Chose a host species with the required adsorptions. 4). Host species compatibility. Some host species may not be compatible with other species in multiple-labeling experiments. In general, all secondary antibodies should come from the same host species for multiple labeling.

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Step 4.   Select secondary antibody specificity under "Antibody Description".

The following explanations of terms may assist in selecting the most appropriate antibody specificity.

Note: Immunoglobulins from different species share similar structures, with similarities being related to closeness in phylogeny. Antibodies against immunoglobulins from one species may cross-react with a number of other species, unless they have been specifically adsorbed against the cross-reacting species. Antibodies that have been adsorbed against other species will contain "(min X...Sr Prot)" in the antibody description.

Anti-Armenian Hamster IgG vs Anti-Syrian Hamster IgG

Most, if not all, hamster monoclonal antibodies are derived from Armenian hamster spleen cell-mouse myeloma hybridomas. The IgG produced by these hybridomas is Armenian (not Syrian) hamster IgG. Most commercially available polyclonal anti-hamster IgG antibodies have been anti-Syrian hamster IgG, which are not as effective as anti-Armenian hamster IgG in detecting Armenian hamster IgG monoclonal antibodies.

Caution:  Anti-Armenian Hamster IgG (H+L) (min X Bov, Hu, Ms, Rb, Rat Sr Prot) may not recognize all Armenian hamster monoclonal antibodies, since it has been adsorbed against closely related species (in bold). Therefore, it is better to use an antibody adsorbed against fewer and/or less closely related species, such as Anti-Armenian Hamster IgG (H+L) (min X Bov Sr Prot), wherever possible, except in those cases where Armenian hamster monoclonals need to be detected in the presence of mouse or rat immunoglobulins.

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Step 5.    Select the desired probe from those listed at the top the table.

For technical information about probes, click here.

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Step 6.   Find the price, size, and code number of the selected antibody by following the row to the right until it intersects the column of the desired probe.

The top numbers in each cell refer to the price per unit size, and the bottom nine-digit number is the catalog code number.

Step 7.   Complete product description for ordering purposes.

For a complete description of the product, please use the following format to avoid mistakes when placing an order. For example, a product with the code number 115-096-072 should be described as follows: