How many aspects should be considered when choosing a secondary antibody?
In a broad sense, secondary antibodies refer to antibodies that specifically react and bind to primary antibodies. In immunological reactions, it is often necessary to select different secondary antibodies for the test. There are more than one antibody to choose from for the detection of any target protein, and there will be different detection schemes in subsequent experiments. Therefore, when selecting a secondary antibody, the type of primary antibody and the requirements of subsequent detection schemes should be considered comprehensively. In general, the selection of a suitable secondary antibody needs to be considered from the following aspects:
【Species source of primary antibody】
The secondary antibody should be from the same species source as the primary antibody used. For example, if your primary antibody is a mouse monoclonal antibody, the secondary antibody should be anti-mouse secondary antibody (goat anti-mouse or rabbit anti-mouse) etc.); if the primary antibody is a rabbit-derived polyclonal antibody prepared from rabbit serum, the corresponding secondary antibody needs to be an anti-rabbit secondary antibody. That is, select the corresponding secondary antibody against the species according to the species source of the primary antibody. Amethyst can provide you with the most complete imported secondary antibodies against different species, including secondary antibodies against mice, rats, rabbits, goats, sheep, humans, guinea pigs, pigs, horses, cattle, chickens, ducks, etc. .
【Class and subtype of primary antibody】
The secondary antibody should match the class or subclass of the primary antibody. This is usually for monoclonal antibodies. Polyclonal antibodies are mainly IgG immunoglobulins, so the corresponding secondary antibodies are anti-IgG antibodies. The types and subtypes of monoclonal antibodies are usually described in the product instructions. If your primary antibody is mouse IgM, then the corresponding secondary antibody should be anti-mouse IgM. If the primary monoclonal antibody is a certain subclass of mouse IgG (IgG1, IgG2a, IgG2b, IgG3), then almost any anti-mouse IgG will bind to it, or you can choose one that specifically targets this subclass Secondary antibody, for example, if your primary antibody is mouse IgG1, then you can choose anti-IgG1 secondary antibody, which is especially suitable for double labeling experiments. If the species/subclass of the primary antibody is unclear, IgG against the corresponding species can be used. Amethyst can provide you with general IgG (H+L) secondary antibodies, or Anti IgM (mu) Antibody that only specifically binds IgM, Anti IgA (alpha-Antibody for IgA), Anti IgE (epsilon) Antibody for IgE, etc. .
【Species source of secondary antibody】
Generally speaking, different species sources are not necessarily related to the quality of the secondary antibody. There is not much difference between the secondary antibody from goat and the secondary antibody from donkey in general experiments. However, in some special experiments, such as double labeling experiments, if one of the primary antibodies is from goat and the other is from mouse, the corresponding secondary antibodies should be anti-goat and anti-mouse respectively. , the secondary antibody cannot be selected from goat or mouse sources. Amethyst has corresponding donkey-derived secondary antibodies, which are very suitable for similar double-labeled immunization experiments.
[Coupling labeling of secondary antibodies]
Generally speaking, the probes coupled to the secondary antibody mainly include enzymes (horseradish peroxidase HRP and alkaline phosphatase AP or its derivatives APAAP, PAP), fluorescent groups (FITC, Rhodamine, Texas Red, PE , Rhodamine, Dylight, etc.), biotin, gold particles. The choice of secondary antibody for the probe depends mainly on the specific experiment. For western blot and ELISA, the most commonly used secondary antibody is enzyme-labeled secondary antibody; while cell or tissue labeling experiments (cell immunochemistry, tissue immunochemistry, flow cytometry) usually use fluorophore-labeled secondary antibodies, immunohistochemistry Horseradish peroxidase or alkaline phosphatase-conjugated secondary antibodies can also be used in the assay. If you want to amplify the detection signal to a greater extent, you can use the Biotin/Avidin detection system. In some fluorescent detection schemes, different fluorescent labels need to be selected; while gold particle-labeled secondary antibodies are more commonly used in immunoelectron microscopy. Amethyst can provide you with all the different types of labeled secondary antibodies mentioned above.
【Whether the secondary antibody has been adsorbed by serum】
In order to reduce the non-specific binding of the secondary antibody to the sample, Amethyst can also provide you with imported secondary antibodies with different serum adsorption. If your detection sample is a protein of human origin, and the primary antibody is a monoclonal antibody of mouse origin, the secondary antibody needs to choose an anti-mouse-derived secondary antibody. If you have high requirements for the specificity of the experiment, then we ask you Anti-mouse-derived secondary antibodies that have been adsorbed by human serum are recommended. This secondary antibody excludes IgG that may have non-specific reactions with human-derived tissues (such as IgG, etc.) due to human serum adsorption, thus reducing non-specific binding. to the minimum.
【Anti-IgG or Anti-IgG, F(ab’)2 fragment】
In some tissues such as thymus, spleen, blood, and hematopoietic cells, lymphocytes, B cells, etc., there are usually more Fc receptors. At this time, it is best to choose Anti-IgG, F(ab') when choosing a secondary antibody 2 Fragment specific secondary antibody, which eliminates the non-specific binding of the Fc part to IgG. The conventional Anti-IgG (H+L) secondary antibody has a binding reaction with both the heavy chain and the light chain of IgG, that is, it can bind to the F(ab')2 fragment of Fc of IgG; since IgG, IgM, and IgA have Conserved light chain domain, so Anti-IgG (H+L) cross-reacts with them. Anti-IgG, F(ab')2 fragment has been adsorbed by the IgG Fc fragment, so it only binds to the F(ab')2 fragment of IgG. However, IgG, IgM, and IgA all have conserved light chain domains, so There is also cross-reactivity with them.
【Anti-IgG (H+L), Anti-IgG (gamma), Anti-IgM (mu), Anti-IgA (alpha)】
In some special experiments, it is only necessary to detect IgG or IgM in the sample. Conventional Anti-IgG (H+L) has a binding reaction with the heavy chain and light chain of IgG, while IgG, IgM, and IgA all have conservative Therefore, it cannot specifically detect certain types of IgG or IgM. At this time, a secondary antibody against a specific type of Ig molecule is needed. Amicate has a comprehensive secondary antibody off-the-shelf product and can provide the most complete secondary antibody product line.
