Antibody selection method in experiments - Database & Sql Blog Articles

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An antibody is a large Y-shaped protein secreted by plasma cells (effector B cells) and used by the immune system to identify and neutralize foreign substances such as bacteria, viruses, and the like. The antibodies used in different experiments are also very different, depending on the type of test application,

Species of the sample,

The structural properties of the sample protein, the type of the antibody host, and the labeling and detection of the antibody select the monoclonal antibody and the secondary antibody that best meet the actual experimental conditions.
1. Analysis of the types of experimental applications General antibody specifications list the types of analysis that the antibody has been tested and validated for use, such as: can be applied to WB IHC ICC ELASA analysis, etc., if the antibody specification does not mention the type of application, not This means that the antibody is not suitable for this type of analytical application, but merely indicates that it has not been verified by such an analytical test. If the antibody is not suitable for some analytical tests, it will be marked on the antibody specification and is not suitable for an analytical test.

2. Species of the species

Antibodies with the same species or cross-reactivity should be selected. Antibodies may cross-react with the same target protein of different species due to their high amino acid sequence homology, if the sample type is not included in the cross-reactivity species of the antibody specification. The table does not mean that the antibody is not suitable for detecting the protein of the species, but merely indicates that the species has not been verified by this antibody detection. The cross-reactivity should be predicted by sequence alignment, and Expasy and NCBI BLAST can be used. Protein homology alignments were performed for different species.

3. 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 cells. The antibody specification generally has a description of the immunogen. If a protein fragment or a particular isoform or a region of the full length of the protein is to be detected, the antibody prepared using the immunogen containing the fragment domain must be selected. 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 is indicated
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 host is more important. For immunohistochemistry, select and sample as much as possible. The primary antibody of different germline species, so as to avoid cross-reaction between the secondary antibody and 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. For the primary antibody of the source, the secondary antibody may be selected to be anti-rabbit IgG coupled with a detection molecule (enzyme, fluorescein, biotin, etc.). 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, however, serum-containing tissue lysates and tissue culture supernatants contain immunoglobulins, IgG is contained in reduced denatured samples, and IgG molecules 50 and 25 are combined in western blot assays. kDa heavy and light chain strips.
5. The secondary antibody should be selected from the same source as the primary antibody used. For example, if your primary antibody is a mouse monoclonal antibody, the secondary antibody is selected as an anti-mouse secondary antibody. It is recommended to check the secondary antibody instructions to ensure that the antibody is suitable for your assay 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 is derived from a different species and the secondary antibody recognizes one of them separately. The secondary antibody specification should describe its origin with other species. Whether the immune ball has cross-adsorption.
7. Fluorescent and chemiluminescent labels are linked to the secondary antibody to detect binding of the antibody. The selection of the label depends on several parameters:
Detection method:
Fluorescent or colored precipitates, which emit visible light when excited by a specific wavelength of light.

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