Second antibody selection method - Database & Sql Blog Articles

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First, the introduction of the secondary antibody:

Secondary Antibodies

:

Broadly speaking

Used

Antibodies that specifically react with and bind to the primary antibody. In immunological reactions, it is often necessary to select different secondary antibodies for the test.

.

There are more than one antibody to choose from to detect any target protein, and there are different detection schemes in subsequent experiments. Therefore, when selecting a secondary antibody, comprehensively consider the type of primary antibody and the requirements of subsequent testing protocols. Say, choosing the right secondary antibody needs to consider the following aspects:

1,

Source of primary antibody

The secondary antibody should be selected from the source of the primary antibody used. For example, the primary antibody is a mouse monoclonal antibody, and the secondary antibody is a secondary antibody against mouse; if the primary antibody is a polyclonal antibody prepared from rabbit serum, the anti-rabbit is used. Secondary Antibodies.

2,

Primary antibody subtype

Second resistance

want

Matches the class or subtype of the primary antibody, usually for monoclonal antibodies. The polyclonal antibody is mainly an IgG-like immunoglobulin, so the corresponding secondary antibody is an anti-IgG antibody. If the primary antibody is mouse IgM, the secondary antibody corresponding to the secondary antibody should be anti-mouse IgM.

3,

Source of secondary antibody

In general, there is no inevitable connection between the source of different species and the quality of the secondary antibody. The secondary antibody derived from goat and the secondary antibody derived from sputum are not much different in the general experiment. However, in the double-label test, One of the primary antibodies is of goat origin and the other is of mouse origin. The corresponding secondary antibodies are resistant to goat and anti-mouse secondary antibodies. At this time, the secondary antibody cannot be selected from goat or mouse.

4,

Secondary antibody coupling marker

In general, probes coupled to secondary antibodies are mainly enzymes, fluorescent groups, biotin, and gold particles. Which secondary antibody to use depends on the specific experiment. For Western blot and ELISA, the most commonly used secondary antibody is the enzyme-labeled secondary antibody; in cell or tissue labeling experiments, fluorophore-labeled secondary antibodies are usually used for immunohistochemistry. Horseradish peroxidase or alkaline phosphatase-labeled secondary antibodies can also be used.

5,

Whether serum has been adsorbed

If your test sample is human-derived, the primary antibody is a mouse-derived monoclonal antibody, and the secondary antibody should be selected against a mouse-derived secondary antibody. If the specificity of the test is high, it is recommended to use human serum. The anti-mouse secondary antibody, which excludes IgG that may react non-specifically with human tissue due to adsorption by human serum, thus minimizing non-specific binding.

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