How to choose an antibody
1. How to store and aliquot antibodies?
For many antibodies, cryopreserving in small aliquots at -20 ℃ or -80 ℃ is the best storage condition. Antibody storage should avoid repeated freezing and thawing, aliquoting can minimize the damage caused by freezing and thawing to the antibody, and it can also avoid the introduction of contamination by removing the antibody from the same tube multiple times. But each serving should not be less than 10 uL. The smaller the aliquoted volume, the more the antibody concentration will be affected by evaporation and tube wall adsorption. In most cases, antibodies can be stored for 1-2 weeks at 4°C. But there are some exceptions. In order to prevent microbial contamination, an appropriate concentration of NaN3 can be added to the antibody. If you want to stain or treat living cells with antibodies, or use antibodies for in vivo studies, you cannot use antibody preparations containing NaN3; this antibacterial agent is toxic to most organisms.
Part of the antibody with glycerol can be stored directly at -20°C.
2. How to choose the primary antibody correctly?
Find the relevant primary antibody product according to the tested species and experimental method, and the nature of the sample determines which antibody is most suitable. The following aspects need to be considered:
(1) The detected protein area. Some antibodies can only recognize reduced and denatured proteins, because this exposes the hidden epitopes in the secondary and tertiary structure formed by protein folding. Some antibodies
can only recognize epitopes on proteins in their natural, folded state. In immunohistochemistry, some antibodies are only applicable to unfixed frozen tissues. If there is no antigen retrieval step to reverse the cross-linking caused by formalin fixation, other antibodies cannot be combined with formalin-fixed and paraffin-embedded tissues. Target binding.
(2) Sample processing method; some antibodies require samples to be processed in a specific way.
(3) The organism producing the primary antibody should be different from the sample organism; this is to avoid cross-reaction between the anti-immunoglobulin secondary antibody and the endogenous immunoglobulin in the sample.
3. How to choose the secondary antibody correctly?
The secondary antibody refers to the antibody that specifically reacts and binds with the primary antibody. In the immunological reaction, it is often necessary to select different secondary antibodies for the test. Consider the following:
(1) A secondary antibody for the primary antibody; for example, if the primary antibody is derived from a mouse, the secondary antibody must be anti-mouse.
(2) The secondary antibody needs to match the category or subclass of the primary antibody; for example, the primary antibody is IgM, and the secondary antibody must be an anti-IgM antibody.
(3) In order to increase the specificity and reduce the background: you can choose the antibody of the Fab segment (with the Fc segment removed), and the secondary antibody adsorbed by the serum of the specimen source species.
(4) Check the instructions of the secondary antibody, and select the secondary antibody that has been verified and can be used in the corresponding experimental method.
(5) The appropriate secondary antibody can be selected through "Related Products" on the primary antibody manual page.
4. Secondary antibody coupling label
The label is coupled to the antibody to show the presence of the target protein. The choice of marker depends on the experimental application. There are:
(1) Fluorescent markers; fluorescent markers emit light in the visible range when excited by light of a specific wavelength. There are a variety of fluorescent markers to choose from, they have unique excitation and emission characteristics.
(2) Enzyme labeling; Enzyme labels such as horseradish peroxidase (HRP) and alkaline phosphatase (AP) form colored precipitates when mixed with appropriate substrates.
(3) Biotin labeling; biotinylated antibodies can be used to amplify the signal, specifically through avidin-biotin-enzyme or fluorescent dye complex or avidin or streptavidin coupled to enzyme or fluorescent dye .
5. How to determine the dilution ratio of the antibody?
If there is a recommended dilution ratio in the manual, please carry out the experiment according to the dilution ratio. However, due to various factors such as specimen type, experimental conditions, operating environment, etc., the recommended concentration is only for reference. The experimenter needs to perform multiple concentration gradient experiments around the recommended concentration to find the best dilution ratio. If the instruction manual does not recommend the dilution ratio, just indicate "Use at an assay dependent dilution", you need to do a lot of preliminary experiments to find the best dilution ratio.