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Location: Home > Information Center > Technical FAQs > Antibody Technology Column > How to select the primary antibody

How to select the primary antibody

Date: 2015-05-03 Author: Leading Biology Click: 3369

1. Analyze the experimental method of the experimental application

Generally, antibody specifications will list the types of assays that the antibody has been tested for, such as: WB, IHC, ICC, ELISA, FCM analysis, etc. If the type of application is not mentioned in the specification, it does not imply that the antibody is not suitable for this type of experiment, it is merely indicating that this anybody has not been verified by the analytical test. In order to avoid the risk of experimentation, when the application method is not mentioned in the manual, you can apply for the test kit and test it before deciding whether to purchase it. If the antibody is not suitable for some analytical tests, it will be marked on the antibody specification that it is not suitable for this analytical test.


2. Analyze the species of the specimen in the experiment

The general antibody instructions list the specimens to which the animal has been tested and validated, for example, the antibody can be used for experiments on specimens of rats, mice, humans, pigs, sheep, etc. That is to say, you may choose the antibodies that are from the same species or can have cross-reaction, the antibodies may cross-react with the same target protein of different species because of high amino acid sequence homology.

If the type of 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 merely mean that the species has not been verified by the antibody test. In order to avoid the risk of experimentation, try not to use antibodies of the species that are not mentioned in the instructions.

Alternatively, cross-reactivity can be predicted by the sequence alignment, and you can use the Expasy and NCBI BLAST to align sequence to perform protein homology from different species. If the result of the comparison proves that the homology is relatively high, it can also be selected.


3. Analyze the structure of the protein in the samples

Analyzing the structural properties of the sample protein helps to select the most appropriate antibody. At least two factors need to be considered in the domain of the sample protein : the antibody is prepared by immunizing the host with various immunogens, including: Full-length proteins, protein fragments, peptides, whole organisms (bacteria) or cells, the antibody instruction generally have a description of the immunogen, if it is intended to detect a protein fragment or a specific isoform or the region of a whole protein, then the antibodies prepared using immunogens containing this fragment domain should be selected. If it is intended to detect the surface proteins of the living cells by FCM flow, it is necessary to select an extracellular domain containing the surface protein to immunize the antibody.

Sample extraction or processing: Some antibodies require the sample to undergo some special treatment, for example, some antibodies only recognize reduced and denatured, epitopes have been exposed to protein samples that are not obstructed by secondary quaternary structure, on the other hand, some antibodies only recognize proteins in their natural 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. And these application sections of the antibody instructions will be indicated.


4. Selection of antibody host species

In general, when a coupled secondary antibody binds to the primary antibody without a conjugate, the species selection of the primary host animal is important. For immunohistochemistry, the primary antibody of the different germline species of the sample is selected as much as possible. Therefore, the secondary antibody is prevented from cross-reacting with the endogenous immunoglobulin of the sample. For example, if the mouse sample protein is detected, the primary antibody of the mouse or rat source should not be selected, and the primary antibody of the rabbit source is preferably selected. An anti-rabbit IgG conjugated with a detection molecule (enzyme, fluorescein, biotin, etc.) can be selected. If we select the conjugated primary antibody, 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, however, serum-containing tissue lysates and tissue culture supernatants contain immunoglobulins, IgG is contained in reduced denatured samples, and IgG molecules 50 and 25 kDa heavy and light chain strips are combined in western blot assays.

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