Step 1.Select
from 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 IgGantibodies 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 fragmentsof antibodies are
generated by pepsin digestion of whole IgG antibodies to remove most 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 kDa. They are used for specific applications, such as to avoid
binding of antibodies to live cells with Fc receptors or 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 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.
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 fragmentsof 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 kDa.
They can be used to block endogenous immunoglobulins on cells, tissues, or
other surfaces, and to block the exposed immunoglobulins in multiple labeling
experiments using primary antibodies from the same species.
In contrast, divalent (whole IgG or F(ab')2
fragment) antibodies should not be used for blocking since they have two
binding sites. After blocking, some of the binding sites would be available to
capture the primary antibody introduced in a subsequent step, resulting in
higher background and/or coincidental labeling.
Step 2. Select
the host species of the primary antibody.
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.
Step
3.Select the host species of the secondary antibody.
Selection of
the host species for a secondary antibody involves many considerations,
including but not limited to: 1) Antibodies from some host species may not be
adsorbed against cross-reacting species of interest. Chose a host species with
the required adsorptions. 2) 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. 3) 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. 4) Personal preference or experience. In our experience there
appears to be no species specific difference in the quality of secondary
antibodies.
Step
4.Select the 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-IgG (H+L)
These
antibodies react with both the heavy and light chains of the IgG molecule, i.e.
with both the Fc and F(ab')2 / Fab portions of IgG (Figure 1).
Anti-IgG (H+L) antibodies also react with other immunoglobulin classes (IgM,
IgA, IgD, IgE) and subclasses since they all share the same light chains
(either kappa or lambda). Anti-IgG (H+L) antibodies have broader epitope
recognition than anti-fragment specific antibodies. They are suggested for all general immunodetection procedures.
-Anti-IgG, Fc/Fcγ fragment specific
These
antibodies react with the Fc portion of the IgG heavy chain. They have been
tested by ELISA and/or absorbed against Fab fragments. In some cases, they are
additionally tested and/or adsorbed to minimize cross-reactivity to IgM and/ or
IgA. In such cases (anti-human, anti-mouse, and anti-rat), they are labeled
"Anti-IgG, Fcγ".
Caution: Anti-IgG,
Fcγ
fragment specific antibodies may not react equally with all monoclonal IgGs.
For an anti-mouse IgG, Fcγ fragment specific antibody with balanced
reactivity to four subclasses of IgG, select goat anti-mouse IgG (subclasses
1+2a+2b+3), Fcγfragment specific (min X Hu, Bov, Rb Sr Prot).
-Anti-Mouse IgG, Fcγ Subclass specific
These
antibodies react with the Fc portion of the heavy chain of individual
subclasses of mouse IgG. They have been tested by ELISA and/or adsorbed to
minimize cross-reactivity to other subclasses, Fab fragments, IgA, IgM, and a
few other species of IgG. Caution: Anti-Mouse
IgG, Fcγ
Subclass specific antibodies react with individual subclasses of mouse IgG. The
extreme specificity of these antibodies may result in reduced epitope
recognition. They are not intended for general labeling of mouse IgG primary
antibodies. For general labeling, select goat anti-mouse IgG (H+L). For heavy
chain specific, use goat anti-mouse IgG (subclasses 1+2a+2b+3), Fcγfragment specific (min
X Hu, Bov, Rb Sr Prot) or goat anti-mouse IgG, Fcγ fragment specific.
-Anti-IgG, F(ab')2 fragment specific
These
antibodies react with the F(ab')2 / Fab portion of IgG. They have
been tested by ELISA and/or adsorbed against Fc fragments. They are not
specific for IgG since they react with light chains, and therefore also react
with other immunoglobulin classes (IgA, IgM, IgD, and IgE) and subclasses
sharing the same light chains.
-(min X ................. Sr Prot)
Secondary
antibodies against one species may cross-react with other species unless they
have been specifically adsorbed against the other species. Antibodies with
"(min X ... Sr Prot)" in the description have been tested and/or
adsorbed against IgG and/or serum proteins of those species indicated in the
parentheses. They are recommended when the 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 recognize some monoclonals poorly. For example, only use anti-mouse IgG
adsorbed against rat IgG to detect a mouse primary antibody in rat tissue which
contains endogenous rat immunoglobulins, or in a multiple labeling application
which includes a rat primary antibody. Use anti-mouse IgG not adsorbed against rat IgG to detect a mouse primary
antibody in the absence of rat immunoglobulins. Two other examples of
antibodies which have diminished epitope recognition after adsorption with
closely-related species are Anti-Rat IgG (min X ... Mouse ... Sr Prot) and
Anti-Armenian Hamster IgG (min X ... Mouse, Rat ... Sr Prot). Refer to "ML
(Multiple Labeling)" below for further information.
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" to emphasize their usefulness in
multiple labeling in addition to single labeling
Anti-Armenian Hamster
IgG vs Anti-Syrian Hamster IgG
Most 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 species, such as Anti-Armenian
Hamster IgG (H+L) (min X Bov Sr Prot), except in those cases where Armenian
hamster monoclonals need to be detected in the presence of mouse or rat
immunoglobulins.
Step 5.Select
the desired probe from those listed at the top the table.
Step6. Find the price,
size, and code number of the selected antibody by following the row 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.
Step7.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:
FITC-conjugated
|
AffiniPure
|
F(ab')2
fragment
|
of Goat
|
Anti-Mouse
|
IgG, F(ab')2
fragment specific
|
A
B
C
D
E
F
(minimal cross-reactivity to
human, bovine, and horse serum proteins)
|
G
A.
Description
of the probe (fluorophore, enzyme, biotin, etc.) if the antibody is
conjugated. If unconjugated, nothing is required here.
B.
AffiniPure
is our trade name for antibodies isolated from antisera by immunoaffinity
chromatography using antigens coupled to agarose beads.
C.
Form of
the antibody - whole IgG, F(ab')2 fragment, or Fab fragment of the
antibody.
D.
Name of
the host species of the antibody.
E.
Name of
the species the antibody is directed against.
F.
Description
of the antibody specificity.
G.
List of
species against which the antibody has been adsorbed to minimize cross-reactivity.
