Primary antibody selection
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:
 Analysis of the type of testing applications l
 Structural properties of protein samples l
 L sample of species
 L antibody host species
 Labeled antibody and detection l
1. Analyze the type of test application
The general antibody instructions list the types of assays that 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 applicable This type of analytical application, but only 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. 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 consider the domain of the sample protein to be tested: the antibody is prepared by immunizing the host with various immunogens, including: Full-length proteins, protein fragments, peptides, whole organisms (eg bacteria) or cells, antibody specifications generally have a description of the immunogen, if it is intended to detect a protein fragment or a specific isoform or a full length of the protein For regions, antibodies prepared using immunogens containing this 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. l
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. On the other hand, some These 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. antibody manual application part marked l
3. Species of the sample <br> Antibodies with the same species or cross-reactivity should be selected. The antibodies may cross-react with the same target protein of different species because of the high amino acid sequence homology, if the sample type is not included. The cross-reactive species table on the antibody specification 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 the antibody detection, and the cross-reaction should be predicted by the sequence alignment method. Expasy and NCBI BLAST can be applied to perform protein homology alignments of different species.
4. Selection of primary antibody host species
In general, when using a secondary antibody that binds to a secondary antibody and a primary antibody without a conjugate, the species selection of the primary host animal is more 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 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.
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