Selection and positioning of secondary antibodies
Step 1: Select antibodies.
Three secondary antibodies: Whole IgG , F( ab&; rsquo;) 2 fragment, Fab fragment. Â
Whole IgG antibodies are suitable for most situations and are the most cost effective. Whole IgG is a complete antibody molecule with an Fc portion and two Fab portions that bind to the antigen (Figure 1, Jackson ImmunoResearch Laboratories Inc website ) .
The F( ab&;quoquo;)2 fragment antibody is a pepsin-digested IgG to remove the Fc portion, leaving two Fab portions of the bound antigen linked by a disulfide bond. F( ab&; quoquo ;) 2 fragment antibodies are used to avoid binding of antibodies to Protain A or G, or to living cells with Fc receptors. If the primary antibody is a complete antibody molecule, it may bind to the Fc receptor regardless of the form of the secondary antibody.
The Fab fragment antibody was obtained by digesting IgG with papain. These antibodies contain only one Fab binding site. They are used to block endogenous immunoglobulins or to block exposed immunoglobulins in experiments using primary antibody multi-labeled from the same species.
The second step: the source of the primary antibody.
In order to correctly select the secondary antibody, you must know the source of the primary antibody. If the primary antibody is of mouse origin, look for &ld;;anti-mouse&;rdquo;.
Step 3: Select the source of the secondary antibody.
Step 4: Select the specificity of the secondary antibody.
  Because immunoglobulins from different species contain similar structures, antibodies against one species may cross-react with other species unless otherwise treated in advance (eg with “ min X Hu, Bov&;hellip; Sr Prot&; " Right cross-reactivity to human and bovine serum proteins) ), after the adsorption treatment of serum proteins in the parentheses (human, bovine) to reduce cross-reactivity). In the selection of secondary antibodies, antibodies against one of the specific adsorption treatments are used only when primary antibodies from two closely related species are present and one of them needs to be detected. Such antibodies may not respond well to all subtypes of IgG . For example, in a tissue sample of a rat containing endogenous immunoglobulin, the anti-mouse IgG adsorbed against the rat is used to detect the primary antibody of the mouse, or one of the rats is used in the multi-labeling experiment. Anti-time. When rat immunoglobulin is not contained, it is preferred to use an anti-mouse secondary antibody that has not been treated with rat IgG to detect the primary antibody of the mouse. Bovine serum albumin (BSA) and milk powder may contain bovine IgG . Secondary antibodies directed against cattle, goats, horses and sheep react with bovine IgG . Therefore, the use of BSA or milk powder to block or dilute these antibodies may result in a significant increase in background staining and a decrease in antibody titer. At this time, it is recommended to use 5% of the normal serum of the secondary antibody source species to block.
Antibodies such as Anti- IgG (H+L) and heavy and light chains of IgG molecules [such as Fc and F ( ab&; rsquo;) 2/Fab fraction ] can be reacted. It can also react with other immunoglobulin families (such as IgM and IgA ) because all immunoglobulins have the same light chain (&;kappa;chain or λchain). Anti- IgG (H+L) recognizes more epitopes than antibodies that are only resistant to fragments.
Antibodies such as Anti- IgG , Fc fragment specific react with the Fc portion of the IgG heavy chain. They passed ELISA assays and/or tests for Fab fragment adsorption. In some cases, additional testing and processing is required to reduce cross-reactivity with IgM or IgA , which is described as < Anti- IgG, Fc &; gamma; fragment specific&; rdquo;. Note: Not all anti- IgG, Fc antibodies and all subtypes have the same IgG reactivity. To find anti-mouse IgG, Fc &; gamma; antibodies with similar IgG subclass abilities, choose Goat Anti-Mouse IgG (subclasses 1+2a+2b+3), Fc &; gamma; fragment specific (min X Hu, Bov, Rb Sr Prot) .
Anti- IgG , Fc &; gamma; Subclass specific antibodies react with the Fc portions of different mouse IgG subtype heavy chains, respectively. Such antibodies are subjected to ELISA and/or adsorption assays for Fab fragments, IgM and other mouse IgG subtypes.
Anti- IgG , F( ab&; rsquo;) 2 fragment specific and F( ab&; rsquo;) 2/Fab partial reaction of IgG . Such antibodies are tested for adsorption by ELISA and/or Fc fragments. Because of their reaction with the light chain, they also react with other types of immunoglobulins (such as IgM and IgA ) that have the same light chain.
(min X&;hellip; Sr Prot) These antibodies have been tested and adsorbed against serum proteins of IgG and parentheses. Such antibodies are recommended when the presence of immunoglobulins from other species may lead to cross-reactivity. However, when selecting antibodies that have been treated for adsorption by similar species, special care should be taken because the number of epitopes they can recognize is greatly reduced and the ability to recognize monoclonal antibodies is weak. Examples of such cases are Anti-Mouse IgG (min X Rat,&;hellip;.Sr Prot), Anti-Rat IgG (min X Mouse, &;hellip; Sr Prot), and Anti- Ramenian Hamster IgG (min X Mouse, Rat, &;hellip;Sr Prot) .
ML ( Multi- Labeling ) : Some antibodies indicate ML to emphasize that they can be used in multi-label experiments in addition to single labeling.
Step 5: Select the desired probe.
Step 6: Specifications, price and catalog number of the selected antibody.
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