Antibody Selection Guide
There are more than one antibody to choose from to detect any target protein of interest. To select the appropriate antibody for narrowing the antibody selection, you need to consider the following factors:
(1) Type of analysis or application
(2) Structural properties of the sample protein
(3) Species of the sample
(4) Types of antibody hosts
(5) Labeling and detection of antibodies
1 Types of application of the analytical test The general antibody specification lists which type of analysis the antibody has been tested for, such as:
Can be applied to WB IHC ICC ELASA analysis, etc. If the type of application not mentioned in the antibody specification does not mean that the antibody is not suitable for this type of analytical application, but only indicates that it has not been verified by such an analytical test, if the antibody is not Applicable to certain analytical tests, it will be marked on the antibody specification and is not suitable for an analytical test.
2 Structural properties of the sample protein Understanding the structural properties of the sample protein helps to select the most appropriate antibody, at least two factors need to be considered
(1). The domain of the sample protein to be tested: The antibody is prepared by immunizing the host with various immunogens, including immunogens: full-length proteins, protein fragments, polypeptides, whole organisms (eg, bacteria). Or the cell, the antibody specification generally has a description of the immunogen. If it is intended to detect a protein fragment or a specific isoform or a certain region of the full length of the protein, it must be prepared using an immunogen containing the fragment domain. antibody. If it is intended to detect the surface proteins of living cells by FACS flow, it is necessary to select an extracellular domain containing the surface protein to immunize the prepared antibody.
(2) Sample extraction or processing: Some antibodies require the sample to undergo some special treatment, for example, many antibodies only recognize reduced and denatured protein samples whose epitopes have been exposed to the secondary quaternary structure, and In contrast, certain antibodies recognize only proteins in a naturally folded state.
When selecting immunohistochemical antibodies, it should be noted that some antibodies recognize only unfixed frozen tissue, while others are suitable for formaldehyde-fixed paraffin-embedded tissues that do not require antigen retrieval and cross-linking. The application part of the antibody specification indicates that the species of the 3 sample should be selected with the same or cross-reactive antibody. The antibody may cross-react with the same target protein of different species due to its high amino acid sequence homology.
If the type of the sample is not included in the cross-reactive species table on the antibody specification, it does not mean that the antibody is not suitable for detecting the protein of the species, but only that the species has not been verified by the antibody detection, and the sequence ratio should be The correct method to predict cross-reactivity, Expasy and NCBI BLAST can be used for protein homology alignment of different species.
4 Selection of primary antibody host species Generally speaking, when using a secondary antibody that binds to a secondary antibody without a conjugate, the species selection of the primary antibody is important. For immunohistochemistry, choose as much as possible for the sample. The primary antibody of the germline species, thereby avoiding cross-reactivity of the secondary antibody with the endogenous immunoglobulin of the sample.
For example, if the mouse sample protein is detected, the primary antibody of mouse or rat source should not be selected. Preferably, the primary antibody of rabbit origin is selected, and the secondary antibody can be coupled with the detection molecule (enzyme, fluorescein, biotin). Etc.) Anti-rabbit IgG. If the primary antibody with conjugate is selected, the above situation is not applicable. Except for immunohistochemistry, other methods for detecting endogenous immunoglobulin-free samples have little effect on the antibody host species, such as IgG-free. Western blotting of cell lysate samples.
Nonetheless, serum-containing tissue lysates and tissue culture supernatants contain immunoglobulins, and IgG is included in the reduced denatured samples, and heavy and light chain bands of 50 and 25 kDa of IgG molecules are combined in western blot assays.
5 Selection of secondary antibody The secondary antibody should be of the same species source as the primary antibody used. For example, if your primary antibody is a monoclonal antibody to a mouse, the secondary antibody is an anti-mouse secondary. It is recommended to check the secondary antibody instructions to ensure that the antibody is suitable for your application. The secondary antibody is typically linked to fluorescein FITC or a luminescent group.
6 Selection of double-stained antibodies Double immunostaining of cell cultures or tissue sections with unconjugated primary antibodies requires that the primary antibody be derived from a different species and that the secondary antibody recognizes one of them separately. The secondary antibody specification should describe its origin with other species. Does the immune ball have cross-adsorption?
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