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Guida selezione anticorpi

Guida alla corretta selezione degli anticorpi secondari adatti alle vostre applicazioni

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

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 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.

 

  igg_fragments.gif

 
F(ab')2 fragments of 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 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 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, Fc
γ 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), Fcγ fragment 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), Fcγ fragment 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.

 
Step 6.   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.

 

 

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:

 

   
 

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.

   

 

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