Jackson ImmunoResearch Europe, Ltd

Selection and Location of Affinity-Purified
Antibodies

Step 1 | Step 2 | Step 3 | Step 4 | Step 5

Technical Service e-mail
tech@jireurope.com tech@jacksonimmuno.com

Step 1. top of page

Select either "Whole Molecules" or "F(ab')₂ Fragments" of the antibody.

Figure 1. Schematic representation of IgG fragments generated by enzymatic digestions.

Figure 1. Schematic representation of IgG fragments generated by enzymatic digestions.

F(ab')₂ fragments (see Figure 1) are used when binding of whole molecule, secondary antibodies to Fc receptors on cell surfaces needs to be avoided. As an alternative, binding of whole molecule, secondary antibodies to Fc receptors may be blocked by incubating cells at 4°C in a buffer containing sodium azide and normal serum from the host species of the secondary antibody.

Please note that if a primary antibody is not an F(ab')₂ fragment, it may also bind to Fc receptors, and blocking with normal serum from the host species of the secondary antibody may not always be successful.

Caution: Never block with normal serum or IgG from the host species of the primary antibody when using a labeled, secondary antibody.)

Step 2. top of page

Find the species of the antigen with which you would like the antibody to react, in the left-hand column labeled "Antibody".

Step 3. top of page

Select the host species in which you want the antibody to be made, under the column labeled "Host".

According to our testing, there appears to be no species specific difference in the quality of antibody conjugates. Therefore selection of the host species should be based on other criteria.

For example, when using Protein A-agarose to separate antigen-antibody complexes from other components, rabbit antibodies should be used since goat IgG, as well as IgG from other species, may not bind as well to Protein A.

Caution: When selecting a secondary antibody, avoid the use of antibodies that have been adsorbed against closely related species, unless it is absolutely necessary to detect one species in the presence of the other. Such antibodies may not react well with all subclasses of IgG, especially those subclasses which are most closely homologous to the species they were adsorbed against. For example, do not use an anti-mouse IgG that has been adsorbed against rat IgG unless you are trying to detect a mouse primary antibody in rat tissue that contains rat immunoglobulin, or in some other tissue in the presence of a rat primary antibody. Conversely, if you wish to detect a mouse primary antibody in the absence of rat immunoglobulins, it is best to use an anti-mouse secondary antibody that has not been adsorbed against rat.

Step 4. top of page

Select an antibody from the column labeled "Antibody".

The following explanations of terms used in this catalog are included to help select the most appropriate antibody from the multiple choices available.

Caution: Antibodies against one species may cross-react with a number of other species, unless they have been specifically adsorbed).

-Anti-IgG (H+L)	This antibody reacts with both the heavy and light chains of the IgG molecule, i.e. it reacts with both the Fc and F(ab')₂ portions of IgG (see Figure 1). Anti-IgG (H+L) also reacts with other immunoglobulin classes (e.g. IgM, IgA, etc.) since all immunoglobulins share the same light chains (either kappa or lambda).
-Anti-IgG, Fc fragment specific	This antibody reacts with the Fc portion of the IgG heavy chain. In each case, it has been adsorbed against F(ab')₂ fragments. In some cases, the antibodies are additionally adsorbed to minimize possible cross-reactivity to IgM and IgA, or to IgM alone. In such cases (anti-human, anti-mouse, and anti-rat), they are labeled "Anti-IgG (Fcγ)". Caution: Anti-IgG (Fc) antibodies do not react to all subclasses of IgG as equally as either Anti-IgG (H+L) antibodies or Anti-IgG, F(ab')₂fragment-specific antibodies.
-Anti-IgG, F(ab')₂ fragment specific	This antibody has been adsorbed against Fc fragments and therefore reacts only with the Fab portion of IgG. Since it reacts with light chains, it also reacts with other immunoglobulins sharing the same light chains.
-(min X ................. Sr Prot)	These antibodies have been tested by ELISA and/or adsorbed against the IgG and serum proteins of those species indicated in the parentheses. They are recommended when the possible presence of immunoglobulins from other species may lead to interfering cross-reactivities. However, caution should be exercised when considering antibodies that have been adsorbed against closely-related species since they have greatly reduced epitope recognition and may recognise some subclasses of IgG very weakly. Two examples of antibodies, which have diminished subclass recognition after adsorption with closely-related species, are Anti-Mouse IgG (min X Rat and other species Sr Prot) and Anti-Rat IgG (min X Mouse and other species Sr Prot). The following abbreviations are used in the parentheses: Bov = Bovine Ck = Chicken Gt = Goat GP = Guinea Pig Sy Hms = Syrian Hamster Hrs = Horse Hu = Human Ms = Mouse Rb = Rabbit Shp = Sheep Sw = Swine Sr = Serum Prot = Protein.
-ML (multiple labeling)	Some antibodies are designated ML since most multiple-labeling experiments require the use of these antibodies to minimize cross-reactivities to other species. However, some of them may also be suitable for single labeling (subject to the caution in Step 3).

Anti-Armenian Hamster IgG vs Anti-Syrian Hamster IgG

Most, if not all, hamster monoclonal antibodies are derived from Armenian hamster-mouse 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 monoclonal antibodies.

Caution: Anti-Armenian Hamster IgG (H+L) (min X Bov, Hu, Ms, Rb, Rat Sr Prot) may not recognise all Armenian hamster monoclonal antibodies, since it has been adsorbed against closely related species. Therefore, it is better to use an antibody adsorbed against fewer 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.

Step 5. top of page

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 refer to the price per unit size and the bottom nine digit number is the code number.

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:

FITC-conjugated \|	AffiniPure	F(ab')₂ fragment \|	of Goat \|	Anti-Mouse \|	IgG, F(ab')₂ fragment specific \|
A		B	C	D	E

(minimal cross-reactivity to human, bovine, and horse serum proteins) \|
F

A.	Description of the probe, if it is conjugated. If unconjugated, nothing is required here.
B.	Form of the antibody - either a whole molecule of the antibody or a F(ab')₂ fragment of the antibody.
C.	Description of the host species.
D.	Description of the species from which the antigen is purified.
E.	Description of the antigen.
F.	Description of species against which the antibody has been adsorbed to minimize cross-reactivity.